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

The effects of WR-1065 (2-((aminopropyl)amino)ethanethiol) on cell cycle progression, Topoisomerase (topo) II alpha activity, and Topo II alpha phosphorylation in Chinese hamster ovary (CHO) cells have been investigated. Exposure of CHO cells to 0.4 microM of WR-1065 for 30 min did not effect cell cycle progression nor Topo II alpha activity and phosphorylation status. However, concentrations ranging from 4 microM to 4 mM were equally effective in significantly altering these three end points. Cell cycle progression was analysed 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 II alpha activity was measured using a kinetoplast DNA (kDNA) decatenation assay. Enzyme activity was reduced by 50% relative to control levels throughout the 4 microM to 4 mM dose range tested. Likewise, Topo II alpha phosphorylation levels, analysed using an immunoprecipitation assay and an antibody specific to the 170 kDa band of Topo II, decreased between 42% to 48% of control levels. Inhibition of Topo II alpha 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.