Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine

Purpose: 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.

Methods: RGC-5 cells were differentiated 24 hr 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 microM) and to high levels of homocysteine (1 mM), glutamate (1 mM), and oxidative stress (25 microM: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.

Results: 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 hr differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine -differentiated RGC-5 cells with 50, 100, or 250 microM 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 the control (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 hr treatment of non-differentiated RGC-5 cells with 5 mM homocysteine decreased survival by 50%.

Conclusions: RGC-5 cells differentiated for 24 hr 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.