Staurosporine-induced differentiation of the RGC-5 cell line leads to apoptosis and cell death at the lowest differentiating concentration.
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.
Centre of Ophthalmology, University Eye Hospital Tübingen, Tübingen, Germany.
Maximillian
M
Schultheiss
Sven
S
Schnichels
Kapka
K
Miteva
Katrin
K
Warstat
Peter
P
Szurman
Martin S
MS
Spitzer
Sophie
S
Van Linthout
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D016923:Cell Death; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation; D002470:Cell Survival; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D016133:Polymerase Chain Reaction; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000473:pathology*; D019311:Staurosporine / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein / Q000235:genetics
D017304:Annexin A5; C494826:BAX protein, human; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D003609:Dactinomycin; C025942:7-aminoactinomycin D; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D019311:Staurosporine