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C. J. Lieven, K. M. Thurber, E. J. Levin, L. A. Levin; Sequence of Apoptosis Signaling Induced by Serum Deprivation in Differentiated RGC-5 Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3192.
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Serum deprivation induces apoptosis in many cell types, but can be prevented by drugs that scavenge or inactivate reactive oxygen species, implicating the latter in apoptosis signaling. Previous work in our laboratory showed that superoxide was an intracellular signal for cell death in primary retinal ganglion cells after axotomy, a condition causing both axonal injury and deprivation of tectum-derived growth factors. We studied superoxide signaling of serum deprivation-induced apoptosis in a transformed retinal ganglion cell line (RGC-5), in order to elucidate whether it was acting as an upstream or downstream signal.
We correlated superoxide levels with the time course of cytochrome c release after serum deprivation. RGC-5 cells were cultured for 24 hrs and differentiated to a neuronal phenotype with staurosporine. Four hours after differentiation, cells were either left undisturbed, switched to a serum-free medium, or treated with etoposide, a known inducer of apoptosis. Untreated and etoposide-treated cells were stained with hydroethidium and fixed after 1 and 24 hours in culture. Serum-deprived and untreated cells were similarly stained and fixed at 24 and 72 hrs post-treatment. Cytochrome c was localized by immunofluorescence. Cells were photographed for quantitative and qualitative analysis. Tests for significance were with Student’s t or chi-square.
Serum deprivation led to a significant increase in superoxide levels in differentiated RGC-5 cells compared to control cells at 72 hrs (1.73 ± 0.14 vs 1.00 ± 0.02; p<0.001). Analysis of cytochrome c localization along with superoxide levels showed that the rise in superoxide preceded the release of cytochrome c from the mitochondrion. Superoxide levels return to basal levels after cytochrome c release, indicating superoxide’s role as an upstream signaling molecule in the apoptotic cascade.
Superoxide production in serum-deprived RGC-5 cells is an early signal in triggering apoptosis. Work is being performed in our laboratory now to determine the sites of action of superoxide, and ways of preventing or reversing its effects before caspase activation can occur and cells become committed to an apoptotic fate.
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