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.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Tamas
T
Kriska
Vladislav V
VV
Levchenko
Fong-Fong
FF
Chu
R Steven
RS
Esworthy
Albert W
AW
Girotti
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