Purchase this article with an account.
M.L. Krause, J.–K. Shen, Y.–J. Chen, X.–R. Yang, M. Swaim, A. Symons, P.A. Campochiaro; Development of a Sensor for Oxidative Stress in Retinal Pigmented Epithelial (RPE) Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2022.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study was to develop a sensor for oxidative stress in RPE cells.
Heme oxygenase–1 (HO–1) is an enzyme that is up–regulated by several types of oxidative stress against which it provides protection. The HO–1 promoter contains an antioxidant response element (ARE) that binds the transcription factor Nrf–2. AREs that differ somewhat in their responsiveness to varioustypes of oxidative stress occur in other genes. Either alone or in combination, 2 repeated sequences of AREs from mouse NQO1, GSTP, GST–ya, or HO–1 were cloned upstream of a 4.5 kb fragment of the human HO–1 promoter coupled to firefly luciferase. These constructs or the control construct pGL2HO–1 were transfected into ARPE19 or HEK293 cells, and after 24 hours the cells were challenged with one of several types of oxidative stress (H202, paraquat, spermine, iron, or hyperoxia). The reporter gene expression level was determined by a dual luciferase reporter gene expression assay.
In ARPE19 cells, transfection of only one fragment, pGL2NQO1ARE–HO–1, resulted in a significant increase in reporter gene expression in response to exposure to H202. These cells also showed increases in reporter gene expression ranging from 3– to 20–fold in a dose–dependent manner after exposure to paraquat, iron, or spermine, but hyperoxia did not cause a significant increase in luciferase levels. HEK293 cells transfected with pGL2NQO1ARE–HO–1 also showed an increase in luciferase expression when exposed to H202, but to a lesser extent than ARPE19 cells.
In RPE cells transfected with a pGL2NQO1–HO1 promoter fragment coupled to a reporter gene, exposure of the cells to various types of oxidative stress results in reporter gene expression. Thus, a pGL2NQO1–HO1 promoter fragment coupled to an antioxidant transgene may provide the basis for oxidative stress–inducible gene therapy.
This PDF is available to Subscribers Only