Abstract
Purpose: :
The acetylation state of histones is modulated by histone deacetylase and histone acetyltransferase, and is an important component in controlling gene transcription, including effects on neuronal differentiation. We previously showed (IOVS, in press) that staurosporine differentiates the RGC–5 cell line into a cell resembling a primary retinal ganglion cell (RGC). We studied the relationship of staurosporine–mediated differentiation with histone acetylation state.
Methods: :
100 µL of cells suspended in RGC–5 growth media were plated into 96–well plates at a density of 5000 cells/mL. At 24 hours after plating, cells were treated with trichostatin A (TSA) and/or staurosporine to final concentrations of: 150 nM (TSA), 316 nM (staurosporine), and 150 nM/50 pM (TSA/staurosporine, respectively). Treatments were performed in sextuplicate. Cells classified as differentiated (more than two processes longer than the soma) from each condition were selected for imaging using NeuronJ. Statistics were performed using 2–sample 2–sided t–tests assuming the variances were unequal.
Results: :
Treatment of the rat retinal ganglion cell precursor line RGC–5 with TSA (150 nM), a potent histone deacetylase inhibitor, caused differentiation. Quantitative differences between TSA– and staurosporine (316 nM)–mediated differentiation included the proportion of cells that were classified as differentiated, and the mean number of primary neurites extending from differentiated cells’ somas (3.0±0.4 (TSA) vs. 5.5±0.7 (staurosporine); p = 0.006). Staurosporine induced differentiation within one day, while TSA mediated differentiation was observed five days after treatment. Simultaneous treatment of cells with TSA and a sub–threshold dosage of staurosporine (50 pM) resulted in no further increase in the proportion of cells that were classified as differentiated compared to TSA alone, but a significant increase in the mean number of primary neurites per differentiated cell (3.0±0.4 (TSA) vs. 7.0±0.7 (TSA/staurosporine); p = 0.002). Combined TSA and staurosporine treatment resulted in an approximately three–fold increase in mean total neurite length per differentiated cell compared to TSA or staurosporine alone. The mean total neurite length per differentiated cell was not observed to be significantly different between the TSA and staurosporine treatment regimes.
Conclusions: :
These data suggest that TSA and staurosporine induce differentiation in RGC–5 cells through different mechanisms, but that at some point there is a common target of interaction, as evidenced by the synergistic action of the two drugs.
Keywords: differentiation • ganglion cells • transcription