Novel enrichment of tumor cell transfectants expressing high levels of type 4 glutathione peroxidase using 7alpha-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, 7alpha-hydroperoxycholesterol (7alpha-OOH), as a selection agent. Cells recovering from a highly toxic dose of liposomal 7alpha-OOH were found to be substantially more resistant to a second 7alpha-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 7alpha-OOH-selected population exhibited uniformly high GPx4 activities (each approximately 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 7alpha-OOH. Compared with conventional "hit or miss" cloning procedures, the 7alpha-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.
Kriska
Tamas
T
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Levchenko
Vladislav V
VV
Chu
Fong-Fong
FF
Esworthy
R Steven
RS
Girotti
Albert W
AW
D002460:Cell Line; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D019076:Transgenes / Q000235:genetics*
D012333:RNA, Messenger; C041011:cholesterol 7-hydroperoxide; D004247:DNA; D002784:Cholesterol; D005979:Glutathione Peroxidase