advertisement
Abstract #25331 Published in IGR 12-1
Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine
Ganapathy P S; Dun Y; Ha Y; Duplantier J; Allen J B; Farooq A; Bozard B R; Smith S BCurrent Eye Research 2010; 35: 80-90
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 24hr using 316nM 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 (mu)M) and to high levels of homocysteine (1mM), glutamate (1mM), and oxidative stress (25 (mu)M: 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 10mM) 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 24hr differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine -differentiated RGC-5 cells with 50, 100, or 250 M 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 1mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18hr treatment of non-differentiated RGC-5 cells with 5mM homocysteine decreased survival by 50%. Conclusions: RGC-5 cells differentiated for 24hr with 316nM 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.
S. B. Smith. Medical College of Georgia, 1459 Laney-Walker Blvd., CB 2820, Augusta, GA 30912-2000, United States. sbsmith@mail.mcg.edu
Classification:
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
11.8 Neuroprotection (Part of: 11 Medical treatment)
