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A. F. Thompson, L. A. Levin; Molecular Specificity of RGC-5 Cell Differentiation by Staurosporine. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3577.
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We previously showed that the RGC-5 cell line can be differentiated with the broad spectrum protein kinase inhibitor staurosporine into retinal ganglion cell- (RGC-) like cells. K252a, K252b, and K252c are kinase inhibitors that are structurally similar to staurosporine. We hypothesized that these molecules would also differentiate RGC-5 cells, but not pheochromocytoma (PC-12 and PC6-3) or fibroblast (3T3) cells.
RGC-5, PC-12, PC6-3, and 3T3 cells were plated in duplicate into 24-well tissue culture plates with appropriate growth media. After 24 hours the cells were treated with staurosporine, K252a, K252b, or K252c at various concentrations. Synergistic or antagonistic effects were analyzed by co-incubation with staurosporine in combination with K252, K252b, or K252c. Cells were photographed and neurites or cell extensions were manually counted by an observer masked to the experimental group.
Treatment of RGC-5 cells with staurosporine caused differentiation, as previously reported. Surprisingly, cells treated with the structurally similar molecule K252a extended fewer neurites than cells treated with staurosporine (1.0 ± 0.3 vs. 3.3 ± 0.2 respectively at 316 nM; p < 0.0001), and did not show typical RGC morphology. K252a caused significantl extension of primary neurites (1.0 ± 0.3 at 316 nM vs. 0.17 ± 0.07 at 0 nM; p < 0.001) but at a level significantly lower than that seen with staurosporine treatment. K252b and K252c had no effect. Similar results were observed with PC-12, PC6-3, and 3T3 cells, in that differentiation was seen with staurosporine but not K252a, K252b, or K252c.
Despite the similar molecular structures, differences between staurosporine and the related compounds K252a, K252b, and K252c are associated with disparate effects on neuronal differentiation significant enough that only staurosporine caused differentiation of RGC-5, PC-12, PC6-3, and 3T3 cell lines. The mechanism of differentiation with staurosporine may be due to a highly specific target of kinase inhibition.
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