GENES REGULATING GLUTATHIONE CONCENTRATIONS IN X-RAY-TRANSFORMED RAT EMBRYO FIBROBLASTS - CHANGES IN GAMMA-GLUTAMYLCYSTEINE SYNTHETASE AND GAMMA-GLUTAMYL-TRANSPEPTIDASE EXPRESSION

The concentration of glutathione (GSH) and the expressions of gamma-gl utamylcysteine synthetase and gamma-glutamyltranspeptidase (GGT) were assessed in rat embryo fibroblasts (REF) displaying various stages of X-ray-induced transformation. A secondary culture of REF cells was irr adiated, and a normal-immortalized cell line (X-REF-23) was isolated. Chronic exposure of X-REF-23 cells to 12-O-tetradecanoylphorbol-13-ace tate (TPA) yielded cells (X-REF-23-TP) capable of benign tumor formati on in nude mice. These cells exhibited GSH concentrations and gamma-gl utamylcysteine synthetase heavy subunit mRNA levels that were similar to 50% less than those measured in X-REF-23 cells. Neither X-REF-23 no r X-REF-23-TP cells exhibited detectable GGT mRNA or activity. Adminis tration of 3 Gy of X-rays followed by chronic TPA treatment yielded ce lls (X-REF-23-TPX) capable of malignant tumor formation in nude mice. These cells expressed GGT mRNA and Concanavalin-A minus GGT activity. One TPX clone (X-REF-23-TPX.1) was chosen for further characterization . Northern blotting of X-REF-23-TPX.1 cells indicated that gamma-gluta mylcysteine synthetase heavy subunit mRNA levels were similar to those of X-REF-TP cells. X-REF-23-TPX.1 cells contained nearly the same amo unt of GSH as X-REF-23 cells. However, the ability of diethylmaleate ( DEM) to deplete GSH was diminished in X-REF-23-TPX.1 cells compared wi th X-REF-23 cells. Furthermore, exposure of X-REF-23-TPX.1 cells to DE M stimulated GSH resynthesis such that the GSH concentration exceeded control values during exposure. The resynthesis of GSH during a DEM ex posure was found to be dependent upon the expression of GGT, as demons trated by inhibition with AT-125. These experiments indicate that ioni zing radiation can lead to elevated constitutive expression of GGT in transformed REF cells and that expression of GGT activity was responsi ble for the increased rate of GSH repletion observed in X-REF-23-TPX.1 cells.