Abstract
Abstract: :
Purpose: External stressors, including oxidants, radiation, toxins, and pathogens, stimulate eukaryotic cells to undergo proliferative and immunogenic responses. Specific genotypic responses to these stressors are controlled by transcription factors, such as NF–kB (RelA/p50 dimer). The cells of the retinal pigment epithelium (RPE) are subject to photo–oxidative stress arising from the interaction of incident light with lipofuscin, melanin, and other pigment granules in the RPE cytoplasm. We have shown that NF–kB is activated in cultured human RPE cells exposed to visible light, and are now examining the modulatory effects of antioxidants on this process. Methods: A line of human–derived RPE cells transfected with the telomerase gene (hTERT–RPE, provided by the Geron Corp., Menlo Park, CA) were grown as previously described (Rambhatla et al., IOVS 43:1622–1630, 2002). These cells develop cytoplasmic melanin pigmentation in culture. The cells were exposed to the continuous wave emission (488.1 nm and 514.5 nm) of an Argon–ion laser. Exposures were made for 10 minutes at 0.5–0.8 W/cm2, a range previously shown to produce photochemical damage in RPE cells. Cells were harvested at various time points up to 24 hrs post–exposure, and NF–kB dimer was detected by an electrophoretic mobility shift assay in nuclear extracts (Mohan & Meltz, Radiat. Res. 140:97–104, 1994). Antioxidant treatment consisted of 20 mM N–acetyl–L–cysteine (NAC), pH 7.4, applied to the cells prior to laser exposure. Results: In the absence of NAC, an increase in nuclear NF–kB commenced at 1 hr post–laser exposure, and declined by 6 hrs. At 24 hrs post–exposure, a second, larger nuclear translocation of NF–kB occurred. Pre–treatment of the cells with 20 mM NAC failed to prevent the nuclear translocation of NF–kB. Conclusions: Photo–oxidative stress induced by visible light exposure caused activation of NF–kB in hTERT–RPE cells. The biphasic pattern of activation observed after photic stress resembled that observed in cells exposed to ionizing radiation. The early increase in nuclear NF–kB was likely due to activation of existing p50/p65, while the late increase represented de novo synthesis. The inability of NAC to reduce nuclear translocation of NF–kB indicates that an NAC–dependent glutathione pathway is not involved in the activation process. It remains to be demonstrated whether other antioxidants (e.g. pyrrolidine dithiocarbamide or ascorbic acid) inhibit NF–kB translocation in RPE cells.
Keywords: transcription factors • retinal pigment epithelium • oxidation/oxidative or free radical damage