Abstract
Abstract: :
Purpose: The RPE is particularly sensitive to laser-induced thermal damage and few treatments are available for ameliorating this type of injury. We investigated thermal preconditioning and Herbimycin A (HA) pretreatments in an in vitro model for their abilities to protect human RPE cells from lethal thermal injury and examined the transcriptional response with cDNA arrays to provide new targets for cytoprotection. Methods: ARPE-19 cells were cultured in DMEM (+10% FCS) in 35 mm dishes and thermally injured by dipping into a 0.9% saline bath at 55° C. Cells treated with 3s/55° C heating followed by a 6h recovery period or HA treated (1.0 ug/ml) for 6h were then subjected to a 9s/55° C exposure. Controls were either not treated or received no pretreatment prior to 9s/55° C heating. For microarray analysis, total RNA from 3s/55° C or HA-treated cultures was harvested 6h later and RNA quality was assessed spectrophotometrically and by 0.9% agarose gel electrophoresis. Total RNA was radiolabeled with 33P and hybridized to GF211 GeneFiltersTM (Research Genetics), which interrogate 4100 human genes. Protein analysis was conducted by immunostaining of western blots. Cell viability was assessed at 24 and 72h after the 9s/55° C treatment by Alamar Blue assay. Results: All untreated cells were dead by 24h following a 9s exposure to 55° C saline. Pretreatment with 3s/55° C heating resulted in 90-100% cell survival when cells were challenged 6h later with lethal 9s/55° C heating. Pretreatment, when compared to controls, with 1.0 ug/ml HA increased cell survival by 80% when cells were challenged by the same 9s/55° C protocol. Inductioin of mRNA transcription (>2.0 fold for each treatment) occurred in common for 8 genes: HSP90, SWI/SNF related matrix associated/actin dependent regulator of chromatin (subfam-a, memb-1), small inducible cytokine A3, crystallin alphaB, FLN29 gene product, claudin 3, and moesin. Protein production for HSP90 and 70 increased in a time-course dependent fashion for both treatments. Thermal preconditioning upregulated 467 genes while HA treatment upregulated 24 genes (>2.0 fold). Prominently suppressed genes for both treatments included transcription factors and kinases. Conclusions: These results suggest that cytoprotection may be due to the contribution of the products of a significant number of other genes in addition to the classic stress response genes, suggesting modulation of these genes might induce thermotolerance and amelioration of thermal injury.
Keywords: gene microarray • retinal pigment epithelium • molecular biology