We have recently reported an innovative approach to use charged fluorochromes such as propidium iodide (PI) in the real-time, dynamic cell viability assays. The present study was designed to provide a mechanistic rationale for the kinetic assays using cell permeability markers. Uptake of PI by live cells, effect on the cell cycle, long term proliferation capacity, DNA damage response and pharmacologic interactions with anticancer drugs were studied using both laser scanning microscopy and laser scanning cytometry. Exposure of human carcinomic alveolar basal epithelial A549 cells in cultures to 1.5 or 7.5 µM of PI for 24 h had minimal effect on cell cycle progression including DNA replication as measured by incorporation of 5’-ethynyl-2-deoxyuridine (EdU) detected by the “click chemistry” approach and measured by laser scanning cytometry. A modest reduction, from 44% to 40% or 33%, in frequency of DNA replicating cells was seen after 48 h at 1.5 or 7.5 µM concentration of PI. There was no evidence of increased phosphorylation of histone γH2AX in cells growing in the presence of 1.5 or 7.5 µM of PI for up to 48 h. Confocal image analysis, of HeLa and NIH 3T3 mouse embryonic fibroblasts growing in the presence of PI showed granular distribution in cell cytoplasm suggesting PI accumulation in endosomes and progressive increase in fluorescence of nucleoli reflecting PI binding to nucleolar RNA. The overall responses of cells to cytotoxic agents were also not affected by the growth in the presence PI. Our data lend further support to the notion that propidium iodide can be effectively used in real-time, kinetic viability assays.

Physiological stress response and oxidative damage are factors for aging processes and, as such, are thought to contribute to neovascular age-related macular degeneration (AMD). Paraoxonase 1 (PON1) is an enzyme that plays an important role in oxidative stress and aging. We investigated association of DNA sequence variants (SNP) within the upstream regulatory region of the PON1 gene with neovascular AMD in 305 patients and 288 controls. Four of the seven tested SNPs (rs705379, rs705381, rs854573, and rs757158) were more frequently found in AMD patients compared to controls (P00.0099, 0.0295, 0.0121, and 0.0256, respectively), and all but one (SNP rs757158) are in linkage disequilibrium. Furthermore, haplotype TGGCCTC conferred protection (odds ratio (OR)00.76, (CI)00.60-0.97) as it was more frequently found in control individ- AGE (2013) uals, while haplotype CGATGCT increased the risk (OR01.55, CI01.09-2.21) for AMD. These results were also reflected when haplotypes for the untranscribed and the 5′untranslated regions (5′UTR) were analyzed separately. To assess haplotype correlation with levels of gene expression, the three SNPs within the 5′UTR were tested in a luciferase reporter assay. In retinal pigment epithelium-derived ARPE19 cells, we were able to measure significant differences in reporter levels, while this was not observed in kidney-derived HEK293 cells. The presence of the risk allele A (SNP rs705381) caused an increase in luciferase activity of approximately twofold. Our data support the view that inflammatory reactions mediated through anti-oxidative activity may be relevant to neovascular age-related macular degeneration.

Results indicated that PON1 is not essential for normal development, function, ageing, and the defense against light damage of the mouse retina. Reduced levels of LPCs in eyes of Pon1(-/-) mice may reflect a decreased activity of phospholipase A2 or altered antioxidative activity in aged eyes.