Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine
Homocysteine is implicated in ganglion cell death associated with glaucoma. To understand mechanisms of homocysteine-induced cell death, we analyzed the sensitivity of the RGC-5 cell line, differentiated using staurosporine, to physiologically-relevant levels of the excitotoxic amino acid homocysteine.
RGC-5 cells were differentiated 24 h using 316 nM staurosporine and tested for expression of Thy 1.2 via immunodetection, RT-PCR and immunoblotting. The sensitivity of staurosporine-differentiated RGC-5 cells to physiological levels of homocysteine (50, 100, 250 µM) and to high levels of homocysteine (1 mM), glutamate (1 mM) and oxidative stress (25 µM:10 mU/ml xanthine:xanthine oxidase) was assessed by TUNEL assay and by immunodetection of cleaved caspase-3. The sensitivity of undifferentiated RGC-5 cells to high (1, 5, and 10 mM) homocysteine was also examined.
Undifferentiated RGC-5 cells express Thy 1.2 mRNA and protein. Staurosporine-differentiated RGC-5 cells extend neurite processes and express Thy 1.2 after 24 h differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine-differentiated RGC-5 cells with 50, 100 or 250µM homocysteine did not alter neurite processes nor induce cell death (detected by TUNEL and active caspase-3) to a level greater than that observed in non-homocysteine-treated, staurosporine-differentiated cells. The 1 mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18 h treatment of non-differentiated RGC-5 cells with 5 mM homocysteine decreased survival by 50%.
RGC-5 cells differentiated for 24 h with 316 nM staurosporine project robust neurite processes and are positive for ganglion cell markers consistent with a more neuronal phenotype than non- staurosporine-differentiated RGC-5 cells. However, concentrations of homocysteine known to induce ganglion cell death in vivo and in primary ganglion cells are not sufficient to induce death of RGC-5 cells, even when they are differentiated with staurosporine.