Abstract
Abstract: :
Purpose: The retinal pigmented epithelium (RPE) is involved in a number of ocular diseases including proliferative vitreoretinopathy (PVR) and age–related macular degeneration (AMD). The human RPE cell line, ARPE–19, allows for assessment of this cell type’s characteristics in both the dividing and differentiated states. p53 tumor suppressor protein is involved in the progression of cell cycle and differentiation of cells. The role of p53 in RPE cells has not been extensively evaluated. Here we use siRNA methods to reduce p53 expression levels in ARPE–19 cells to allow for the evaluation of p53's role in dividing and differentiated cells. Methods: Four siRNA sequences targeting the p53 gene were cloned into Tranzyme's proprietary lentiviral gene transfer vector, TranzVectorTM under the U6 promoter. Constructs were confirmed by sequencing and viral stocks were prepared. ARPE–19 cells were transduced with virus particles containing a p53 siRNA construct. The decrease in p53 levels was assessed by RT–PCR and Western blot. Subsequent experiments used two chemical insults– camptothecin, a topoisomerase I inhibitor known to cause DNA damage, and menadione, a free radical generating agent, to evaluate the effect of reduced levels of p53 on several endpoints including survival, expression of p21/waf–1 and effect on the p21/waf–1 damage response. Results: TranzVector was capable of generating consistent and stable expression of the p53 siRNAs in ARPE cells. RT–PCR and Western blot experiments demonstrated that the siRNAs were capable of significantly reducing p53 levels. As expected, the silencing of p53 led to a decrease of p21/waf–1 transcription in proliferating and differentiated cells. DNA damage induced by camptothecin increased p53 transactivation of p21/waf–1 in proliferating cells, whereas damage from menadione did not. In proliferating cells, p53 silencing reduced cell death induced by camptothecin, but not by menadione. Untransduced differentiating cells were resistant to the same doses of camptothecin and menadione, but were sensitive to higher doses of menadione. Studies of silencing of p53 in the differentiating cells and its' effect on cell survival are in progress. Conclusions: The TranzVector system generated long term, stable expression of p53 siRNAs in the ARPE–19 cells. These experiments have allowed the evaluation of the role of p53 in the response of dividing and differentiating RPE cells to lethal agents. They have demonstrated that a decrease in p53 can have significant impact on how these cells deal with environmental damage.
Keywords: retinal pigment epithelium • cell death/apoptosis • proliferation