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 cyc le progression, topoisomerase (topo) II alpha activity, and topo II al pha phosphorylation in Chinese hamster ovary (CHO) cells have been inv estigated. Exposure of CHO cells to 0.4 mu M of WR-1065 for 30 min did not effect cell cycle progression nor topo II alpha activity and phos phorylation status. However, concentrations ranging from 4 mu M to 4 m M were equally effective in significantly altering these three end poi nts. 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 ac cumulation of cells in G(2) Topo II alpha activity was measured using a kinetoplast DNA (kDNA) decatenation assay. Enzyme activity was reduc ed by 50% relative to control levels throughout the 4 mu M to 4 mM dos e range tested. Likewise, topo II alpha phosphorylation levels, analys ed using an immunoprecipitation assay and an antibody specific to the 170 kDa band of topo II, decreased between 42% to 48% of control level s. Inhibition of topo II alpha activity in cells exposed to WR-1065 is consistent with the associated observation of WR-1065 mediated cell c ycle progression delay and build-up of cells in the G(2) phase of the cell cycle.