949 Differential effects of ifosfamide on lymphocytes subsets correlate with glutathione metabolism and cystine uptake

We used human peripheral blood lymphocytes (PBL) as a model system for investigations of cytostatic drugs on human cells in vitro . We studied the influence of the activated alkylating agent ifosfamide (4-OH-1F) on CD3 + cytotoxic T lymphocytes (CTL) and CD3 + natural killer (NK) cells. Exposure of the cells to 4-OH-1F reduces significantly the cytolytic activity of CTL but less effective the cytolytic activity of NK cells. This correlates with the ability of ifosfamide to decrease differentially the intracellular glutathione (GSH) levels of the two cell types. Analysis of the initial GSH levels of CTL and NK cells of a panel of HLA different blood donors shows that NK cells have significant higher levels of GSH compared to CTL (36.5 ± 7.7 vs 27.2 ± 5.8 nmol GSH/mg protein). However, this difference in initial GSH level in NK cells compared to CTL is about 1.5-fold, whereas the resistance against an 4-oH-IF treatment that leads to an equal GSH depletion in both cell types is about 4-fold. For further analysis this discrepancy we determined the relative rate of GSH synthesis in NK cells and CTL. We could show that NK cells have an about 4-fold greater capacity of GSH synthesis compared to CTL. The synthesis of GSH in lymphocytes is rate limited by the uptake of the amino acid cysteine and its disulfide form cystine. Analysis of transport systems show that NK cells take up cystine very well, but CTL lack this transport system. Cysteine can be taken up by both cell types, but under physiological conditions the extracellular concentrations of cysteine compared to cystine are quite low. However, cystine uptake in CTL can be achieved by addition of thiol compounds, e.g. 2-mercaptoethanesulfonate (mesna), to the medium of the cells. Our data suggest that the GSH levels as well as GSH synthesis of NK cells compared to CTL is related to the difference in the transport system for the amino acid cystine. This work was supported by grant M90/91-Is1 from the Deutsche Krebshilfe and by grant Is31/3 from the Deutsche Forschungsgemeinschaft.