Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine.
OBJECTIVE
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.
Ganapathy
Preethi S
PS
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000, USA.
Dun
Ying
Y
Ha
Yonju
Y
Duplantier
Jennifer
J
Allen
John Bradley
JB
Farooq
Amina
A
Bozard
B Renee
BR
Smith
Sylvia B
SB
D053148:Caspase 3 / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D018698:Glutamic Acid / Q000494:pharmacology; D006710:Homocysteine / Q000494:pharmacology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology*; D018800:Thy-1 Antigens / Q000235:genetics / Q000378:metabolism; D019820:Xanthine / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000494:pharmacology
D012333:RNA, Messenger; D018800:Thy-1 Antigens; D006710:Homocysteine; D019820:Xanthine; D018698:Glutamic Acid; D014969:Xanthine Oxidase; D053148:Caspase 3; D019311:Staurosporine