WR-1065, an active metabolite of the cytoprotector amifostine, affects phosphorylation of topoisomerase IIα leading to changes in enzyme activity and cell cycle progression in CHO AA8 cells

Abstract

The effects of WR-1065 (2-((aminopropyl)amino)ethanethiol) on cell cycle progression, topoisomerase (topo) IIα activity, and topo IIα phosphorylation in Chinese hamster ovary (CHO) cells have been investigated. Exposure of CHO cells to 0.4 μM of WR-1065 for 30 min did not effect cell cycle progression nor topo IIα activity and phosphorylation status. However, concentrations ranging from 4 μM to 4 mM were equally effective in significantly altering these three end points. Cell cycle progression was analyzed by flow cytometry. Following a 30 min exposure to this range of concentrations, cells redistributed throughout the cell cycle with the most prominent changes being an accumulation of cells in G2. Topo IIα activity was measured using a kinetoplast DNA (kDNA) decatenation assay. Enzyme activity was reduced by 50% relative to control levels throughout the 4 μM to 4 mM dose range tested. Likewise, topo IIα phosphorylation levels, analyzed using an immunoprecipitation assay and an antibody specific to the 170 kDNA band of topo II, decreased between 42% to 48% of control levels. Inhibition of topo IIα activity in cells exposed to WR-1065 is consistent with the associated observation of WR-1065 mediated cell cycle progression delay and build-up of cells in the G2 phase of the cell cycle.