Rationale for the real-time and dynamic cell death assays using propidium iodide.

Abstract

We have recently reported an innovative approach to use charged fluorochromes such as propidium iodide (PI) in the real-time, dynamic cell viability assays. This 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 microM 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 microM concentration of PI. There was no evidence of increased phosphorylation of histone gammaH2AX in cells growing in the presence of 1.5 or 7.5 microM 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 PI can be effectively used in real-time, kinetic viability assays.