Novel enrichment of tumor cell transfectants expressing high levels of type 4 glutathione peroxidase using 7α-hydroperoxycholesterol as a selection agent

A novel approach for selecting high expressing cells out of a general population that had been transfected with a construct encoding cytosolic type 4 glutathione peroxidase (GPx4) is reported. The approach is described for GPx4-null COH-BR1 breast tumor cells and is based on use of a highly specific GPx4 substrate, 7α-hydroperoxycholesterol (7α-OOH), as a selection agent. Cells recovering from a highly toxic dose of liposomal 7α-OOH were found to be substantially more resistant to a second 7α-OOH challenge than cells recovering from a less toxic dose, but were much less resistant to t-butyl hydroperoxide (t-BuOOH) or H2O2. Several clones isolated from the general transfectant population exhibited variable, relatively low GPx4 activities. However, clones from the 7α-OOH-selected population exhibited uniformly high GPx4 activities (each ∼3-fold higher than that of the starting transfectant population) and elevated steady-state mRNA levels. t-BuOOH could also be used for selecting high GPx4-expressing cells, but consistent recovery from toxic doses was more difficult than with 7α-OOH. Compared with conventional “hit or miss” cloning procedures, the 7α-OOH approach we describe affords a uniform population of high GPx4-activity cells in a relatively rapid manner. This approach should prove valuable for investigators interested in the peroxide regulatory properties of GPx4, in the context of both cytoprotection and redox signaling.