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Agnieszka Gruszka, Gesine Attrodt, Jose Hurst, Johanna Hofmann, Karl Ulrich Bartz-Schmidt, Martin Stephan Spitzer, Sven Schnichels; The RGC-5 vs. the 661W cell line: similarities, differences & remaining opportunities . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2448.
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© ARVO (1962-2015); The Authors (2016-present)
The RGC-5 cell line is still widely used in retinal ganglion cell (RGC) research although it is well known that this cell line has been dedifferentiated over the years and questions about its origin were raised. The photoreceptor 661W cell line is sharing several similarities and the possibility that the RGC-5 cell line was contaminated with 661W cells. Over the last years different ways to induce redifferentiation were proposed including treatment with Trichostatin A (TSA). The aim of this study was to clarify whether both cell lines are the same and undergo comparable changes when treated with TSA under the same conditions.
RGC-5 & 661W cells were treated for 24, 48, 72, 96 & 120h with TSA according to published protocols (Schwechter 2007 & Wood 2010) to cause redifferentiation. Cell morphology was investigated. Cell viability was analyzed by MTS assay. For apoptosis detection Caspase 3/7-assays were performed and the ratios of cleaved caspase 3/caspase 3 and BAX/Bcl-2 were measured via Western Blot (WB). The expression profile of neuronal & RGC markers such as MAP-2, tau & βIII-tubulin was analyzed by immunostaining (IS). βIII-tubulin expression was detected via WB and Thy-1 expression was shown with a rt-PCR.
TSA treated RGC-5 cells in general appeared with round somata and various neuritic outgrows while 661W cells remained long shaped with less neuritis. Cell viability of TSA treated RGC-5 cells reached a peak after 120h of incubation. This was not observed for the 661W cell line. Caspase 3/7 activity was more influenced in 661W cells and was mostly higher than in RGC-5 cells. In contrast, the cleaved caspase 3/caspase 3 ratio of the 661W cells did not react as strongly as RGC-5 cells but also increased. The expression of βIII-tubulin and Thy-1- significantly increased in RGC-5 cells after long-term incubation with TSA whereas the expression of both in 661W cells did not differ from controls. This finding was also confirmed by IS against βIII-tubulin.
Although the 661W and RGC-5 cell line showed some similarities, with the majority of the methods the reaction differed. Especially the higher expression of the RGC specific markers βIII-tubulin and Thy-1 in the RGC-5 cells proofs that these cell lines cannot be considered as the same. In conclusion, there is a chance that the RGC-5 cells can still be used -under limited conditions- and with a good differentiation protocol.
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