PURPOSE: To detail, by DNA microarrays and cellular structure labeling, the in vitro responses of retinal pigment epithelial (RPE) cells to a nonlethal dose of the oxidant agent hydroquinone (HQ). METHODS: The viability of growth-quiescent ARPE-19 cells after treatment with HQ was measured by XTT conversion, (3)H-leucine incorporation, trypan blue exclusion, and the presence of DNA laddering. The effect of a nonlethal dose of HQ on the localization of apoptosis-induced factor (AIF) and phosphorylation of stress-activated kinase-2/p38 (SAPK2/p38) was detected by immunocytochemistry. Actin structures were visualized by phalloidin staining. Cell membrane blebbing was detected using GFP-membrane-labeled RPE cells (ARPE-GFP-c'-rRas). Changes in gene expression patterns of RPE cells within 48 hours of prolonged treatment with a nonlethal dose of HQ were evaluated by microarray analysis and confirmed by Northern blotting. RESULTS: The viability of RPE after a prolonged sublethal injury dose of HQ was determined by multiple assays and confirmed by the absence of AIF translocation or DNA laddering. Prolonged exposure (16 hours) of RPE cells to a nonlethal dose of HQ resulted in actin rearrangement into globular aggregates and cell membrane blebbing. Kinetic microarray analysis at several time points over a 48-hour recovery period revealed significant upregulation of genes involved in ameliorating the oxidative stress, chaperone proteins, anti-apoptotic factors, and DNA repair factors, and downregulation of pro-apoptotic genes. Genes involved in extracellular matrix functions were also dysregulated. Recovery of RPE cells after the injury was confirmed by the normalization of gene expression dysregulation back to baseline levels within 48 hours. CONCLUSIONS: RPE cells avoided cell death and recovered from prolonged oxidative injury by activating a host of defense mechanisms while simultaneously triggering genes and cellular responses that may be involved in RPE disease development.
PURPOSE: To detail, by DNA microarrays and cellular structure labeling, the in vitro responses of retinal pigment epithelial (RPE) cells to a nonlethal dose of the oxidant agent hydroquinone (HQ). METHODS: The viability of growth-quiescent ARPE-19 cells after treatment with HQ was measured by XTT conversion, (3)H-leucine incorporation, trypan blue exclusion, and the presence of DNA laddering. The effect of a nonlethal dose of HQ on the localization of apoptosis-induced factor (AIF) and phosphorylation of stress-activated kinase-2/p38 (SAPK2/p38) was detected by immunocytochemistry. Actin structures were visualized by phalloidin staining. Cell membrane blebbing was detected using GFP-membrane-labeled RPE cells (ARPE-GFP-c'-rRas). Changes in gene expression patterns of RPE cells within 48 hours of prolonged treatment with a nonlethal dose of HQ were evaluated by microarray analysis and confirmed by Northern blotting. RESULTS: The viability of RPE after a prolonged sublethal injury dose of HQ was determined by multiple assays and confirmed by the absence of AIF translocation or DNA laddering. Prolonged exposure (16 hours) of RPE cells to a nonlethal dose of HQ resulted in actin rearrangement into globular aggregates and cell membrane blebbing. Kinetic microarray analysis at several time points over a 48-hour recovery period revealed significant upregulation of genes involved in ameliorating the oxidative stress, chaperone proteins, anti-apoptotic factors, and DNA repair factors, and downregulation of pro-apoptotic genes. Genes involved in extracellular matrix functions were also dysregulated. Recovery of RPE cells after the injury was confirmed by the normalization of gene expression dysregulation back to baseline levels within 48 hours. CONCLUSIONS: RPE cells avoided cell death and recovered from prolonged oxidative injury by activating a host of defense mechanisms while simultaneously triggering genes and cellular responses that may be involved in RPE disease development.
Authors: Oscar Alcazar; Adam M Hawkridge; Timothy S Collier; Scott W Cousins; Sanjoy K Bhattacharya; David C Muddiman; Maria E Marin-Castano Journal: Mol Cell Proteomics Date: 2009-06-29 Impact factor: 5.911
Authors: Ping Yang; Peter Baciu; Brittany C Parker Kerrigan; Menna Etheridge; Eric Sung; Brett A Toimil; Jacob E Berchuck; Glenn J Jaffe Journal: Invest Ophthalmol Vis Sci Date: 2014-05-06 Impact factor: 4.799
Authors: Astrid Subrizi; Elisa Toropainen; Eva Ramsay; Anu J Airaksinen; Kai Kaarniranta; Arto Urtti Journal: Pharm Res Date: 2014-07-17 Impact factor: 4.200