Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways.
OBJECTIVE
Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium.


METHODS
RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10% fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-kappaB binding activity by electrophoretic mobility gel shift assays (EMSA) and mitochondrial damage by JC-1 (5,5', 6,6'-tetrachloro 1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c.


RESULTS
Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, -8, and -9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-kappaB (NF-kappaB) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells.


CONCLUSIONS
Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.
Charles
Irma
I
Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, Texas, USA.
Khalyfa
Abdelnaby
A
Kumar
D Maneesh
DM
Krishnamoorthy
Raghu R
RR
Roque
Rouel S
RS
Cooper
Nigel
N
Agarwal
Neeraj
N
D000818:Animals; D017209:Apoptosis; D001562:Benzimidazoles / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002461:Cell Line, Transformed; D016895:Culture Media, Serum-Free; D045304:Cytochromes c / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015151:Immunoblotting; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D001562:Benzimidazoles; D002232:Carbocyanines; D016895:Culture Media, Serum-Free; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D045304:Cytochromes c; D020169:Caspases