Mouse embryonic stem (ES) glucose-6-phosphate (G6P) dehydrogenase-deleted cells (G6pdΔ), obtained by transient Cre recombinase expression in a G6pd-loxed cell line, are unable to produce G6P dehydrogenase (G6PD) protein (EC 220.127.116.11). These G6pdΔ cells proliferate in vitro without special requirements but are extremely sensitive to oxidative stress. Under normal growth conditions, ES G6pdΔ cells show a high ratio of NADPH to NADP+ and a normal intracellular level of GSH. In the presence of the thiol scavenger oxidant, azodicarboxylic acid bis[dimethylamide], at concentrations lethal for G6pdΔ but not for wild-type ES cells, NADPH and GSH in G6pdΔ cells dramatically shift to their oxidized forms. In contrast, wild-type ES cells are able to increase rapidly and intensely the activity of the pentose-phosphate pathway in response to the oxidant. This process, mediated by the [NADPH]/[NADP+] ratio, does not occur in G6pdΔ cells. G6PD has been generally considered essential for providing NADPH-reducing power. We now find that other reactions provide the cell with a large fraction of NADPH under non-stress conditions, whereas G6PD is the only NADPH-producing enzyme activated in response to oxidative stress, which can act as a guardian of the cell redox potential. Moreover, bacterial G6PD can substitute for the human enzyme, strongly suggesting that a relatively simple mechanism of enzyme kinetics underlies this phenomenon.
Abbreviations used: diamide, azodicarboxylic acid bis[dimethylamide]; ES, embryonic stem; G6P, glucose 6-phosphate; G6PD, G6P dehydrogenase; G6pdΔ, G6pd-deleted cells; H6PD, hexose-6-phosphate dehydrogenase; Hprt, hypoxanthine—guanine phosphoribosyltransferase; IDH, isocitric dehydrogenase; Mod1, ‘malic’ enzyme; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt]; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NEO cassette, gene encoding neomycin phosphotransferase gene under the control of the phosphoglycerokinase promoter; 6PGD, 6 phospho-gluconate dehydrogenase; PPP, pentose-phosphate pathway; RBC, red blood cells; RT, reverse transcriptase.