title: Promotion of cystine uptake, increase of glutathione biosynthesis, and modulation of glutathione status by S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721) in Chinese hamster cells.
abstract: We recently found that exposure of cells to different aminothiols promotes cystine uptake and leads to an increase of cellular glutathione by new biosynthesis (Issels et al., Biochem. Pharmacol., 37: 881-888, 1988). Therefore, we further investigated whether the known radioprotective and chemoprotective aminothiol derivative S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) or its dephosphorylated form (WR-1065) will lead to similar effects. In order to convert WR-2721 to the free thiol compound (WR-1065) in vitro, the medium also contained 20 U/ml alkaline phosphatase (AP). For uptake studies a modified McCoy's 5A medium supplemented with 0.1 mM [35S]cystine was used. In Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa) cells, WR-2721 exposure alone did not increase the cystine uptake relative to that of control (untreated) cells, while WR-2721 + AP enhanced the uptake of cystine more than twofold in both cell lines. The increase of cystine uptake was dependent on the time of exposure (0-60 min) and the concentrations of WR-2721 (0-8 mM) + AP. Half-maximal uptake of cystine was observed at concentrations of 0.69 and 0.57 mM WR-2721 in CHO and OvCa cells, respectively. Determination of both reduced (GSH) and oxidized (GSSG) cellular glutathione levels after the exposure (0-300 min) to WR-2721 + AP in CHO cells showed a depletion of GSH to less than 10% of the pretreatment value and a 4-fold reduction of the GSH/GSSG ratio. In contrast, in OvCa cells the amount of total glutathione rather increased with no significant change of the GSH/GSSG ratio by the exposure to WR-2721 + AP. Further analysis using high-performance liquid chromatography of cell extracts revealed that the relative amount of incorporated [35S]-cystine into glutathione was increased similarly in both cell lines. The data show that precursor availability and new biosynthesis of glutathione is enhanced by the exposure to WR-2721 + AP in vitro despite the differential modulation of the cellular glutathione status in the two cell lines. These findings may have important implications for the use of aminothiols like WR-2721 in various cells and tissues in regard of their response to chemotherapeutic agents, ionizing radiation and/or hyperthermia.
authors.affiliation: Gesellschaft für Strahlen- und Umweltforschung (GSF), Ludwig-Maximilians-Universität München, West Germany.
authors.forename: R D
authors.initials: RD
authors.lastname: Issels
authors.forename: A
authors.initials: A
authors.lastname: Nagele
mesh_terms: D000469:Alkaline Phosphatase / Q000494:pharmacology; D004999:Amifostine / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003553:Cystine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000032:analysis / Q000378:metabolism*; D009946:Organothiophosphorus Compounds / Q000494:pharmacology*
chemical_list: D009946:Organothiophosphorus Compounds; D003553:Cystine; D000469:Alkaline Phosphatase; D005978:Glutathione; D004999:Amifostine