Purchase this article with an account.
C.J. Lieven, L.A. Levin; Induction of Apoptosis in Differentiated RGC–5 Cells Reduces Expression Levels of Retinal Ganglion Cell Markers . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5497.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We have previously shown that the retinal ganglion cell (RGC)–like RGC–5 line can be differentiated by low concentrations of staurosporine (SS), and that expression levels of some ganglion cell markers are increased by this change (IOVS, in press). As staurosporine is known to initiate cell death via apoptosis at higher concentrations, we examined whether induction of cell death after SS–induced differentiation increased expression of RGC markers.
RGC–5 cells were plated on 100 mm tissue culture plates and allowed to grow for 24 hours before differentiation was induced with staurosporine. After 48 hours in the presence of SS, the cells were treated with rotenone, ionomycin, or left untreated for 6 hours. Undifferentiated, untreated RGC–5 cells were grown in parallel and used as a reference for normal expression levels. After 6 hours, the cells were rinsed with phosphate–buffered saline, and proteins were harvested by standard methods. The proteins were then reduced, run out on a 4–12% polyacrylamide gel, and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies to Thy–1, a ganglion surface marker, and microtubule–associated protein 2 (MAP2), followed by rinsing and exposure to appropriate secondary antibodies. Membranes were treated with ECL solution and exposed for varying lengths of time. Blots were then stripped, and reprobed for actin to verify equal protein loading between samples. Expression levels were quantified by scanning the resulting films and calculating intensity compared to background using NIH ImageJ software.
Treatment with rotenone and ionomycin did not lead to increased expression of MAP2. Rather, induction of cell death with these compounds lead to decreased levels of MAP2 expression (decrease of 87% and 73% compared to untreated, differentiated for rotenone and ionomycin, respectively).
Induction of cell death via other mechanisms does increase expression levels of MAP2 after differentiation with SS, and actually leads to decreased levels of this dendritic marker. This result suggests that the increased expression of RGC markers is not dependent on the shared apoptotic pathway induced by high levels of staurosporine, consistent with our prior findings that inhibition of the apoptotic cascade did not prevent staurosporine–induced differentiation of RGC–5 cells.
This PDF is available to Subscribers Only