Anzeige
Erschienen in:
01.08.2012 | Glaucoma
Staurosporine-induced differentiation of the RGC-5 cell line leads to apoptosis and cell death at the lowest differentiating concentration
Erschienen in: Graefe's Archive for Clinical and Experimental Ophthalmology | Ausgabe 8/2012
Einloggen, um Zugang zu erhaltenAbstract
Background
Supplementation of staurosporine is the method of choice for differentiating the solely existing retinal ganglion cell (RGC)-5 cell line. This differentiation was initially claimed to be in the absence of apoptosis, but some publications supposed the induction of apoptosis during staurosporine induced RGC-5 differentiation. In respect to these inconsistencies in the literature, we investigated in detail whether RGC-5 cell differentiation by staurosporine induces apoptosis or not.
Methods
Amounts of 50 nM, 200 nM, 300 nM, and 600 nM of staurosporine were supplemented on RGC-5 cells for 24 h. Cell morphology and cell death, via propidium iodide staining, were evaluated 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 quotient of Bax to Bcl-2 mRNA expression via caspase 3/7 activity assay, flow cytometry, and real-time PCR, respectively.
Results
RGC-5 cells started to change their morphology and their expression of neuronal markers at 50 nM of staurosporine. This was associated with apoptosis and cell death, as 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, and a 12–fold (p < 0.0005) increase in propidium iodide positive cells, respectively. Furthermore, staurosporine led to a dose-dependent increase in apoptosis and reduction in cell viability, cell density, and cell proliferation.
Conclusions
The lowest staurosporine concentration inducing RGC-5 cell differentiation is accompanied by apoptosis and cell death.