Role of p53 in Nitric Oxide–induced Apoptosis in the MCMV Infected Murine RPE Cells

Abstract

Abstract: : Purpose:The goal of these studies was to investigate nitric oxide (NO) production in MCMV infected murine retinal pigment epithelial (RPE) cells and the effect of released NO on these cells. In addition, the role of p53 protein in NO–induced apoptosis in the cells was studied. Methods:Primary RPE cell cultures were established from C57B/6 mice, and confluent RPE cultures were infected with MCMV (Smith strain) with or without daily treatment of aminoguanidine (AG), a selective iNOS inhibitor. The conditioned medium was collected for measurement of the level of nitrite by a fluorometric assay with DAN reagent at 2, 3, 5, and 7 days after inoculation. Apoptosis in the RPE cells was assessed for fragmentation of DNA by TUNEL labeling at different time points and the expression of p53 protein in the RPE cells was evaluated by Western blot. Results:Fluorometric DAN analysis showed a significant increase in NO production in MCMV infected murine RPE cells when compared with time matched uninfected cells or with AG treated cells. The MCMV infected murine RPE cells with high level of NO showed an increased number of apoptotic cells and increased expression of p53 protein. Conclusions:MCMV Infection of murine RPE cells results in increased NO production and increased NO production correlates with an increase in the number of apoptotic RPE cells suggesting that apoptosis observed during MCMV retinitis in vivo may be attributable to NO production by RPE and perhaps by other retinal cells. These results also suggest that NO–induced apoptosis of cultured murine RPE cells may require upregulation of p53.

Keywords: cytomegalovirus • retinitis • apoptosis/cell death