The induction of hsp-70 by gamma irradiation was studied in Chinese hamster ovary (CHO) cells. Synthesis of hsp-70 was assessed by SDS-PAGE of radiolabeled proteins while levels of hsp-70 mRNA were estimated by Northern blot analysis of total RNA. SDS-PAGE analysis indicated that the synthesis of hsp-70 was enhanced in cells exposed to 400 or 1000 Gy. Northern blot analysis of RNA from cells exposed to 1000 Gy indicated a rapid increase in mRNA capable of hybridizing to an hsp-70 oligonucleotide probe. These experiments represent the first report demonstrating that ionizing radiation can increase hsp-70 mRNA.

The relationship between glutathione metabolism, menadione sodium bisulphite oxidation of protein thiols, and the synthesis of hsc70 was investigated using CHO cells. A 30-min/37 degrees C exposure to menadione, a compound which redox cycles to produce superoxide anion radicals and hydrogen peroxide, resulted in rapid accumulation of hsc70 mRNA. PAGE and Western blot analysis indicated increased synthesis such that accumulation of hsc70 occurred. These changes were preceded by rapid oxidation of GSH to GSSG, followed by GSH depletion, and subsequent protein thiol oxidation. As a test of whether a correlation existed between GSH oxidation and depletion, protein thiol oxidation and hsp synthesis, cells were exposed to menadione in the absence and presence of glucose. Synthesis of hsc70 was increased in cells exposed to menadione in the absence of glucose compared with its presence. As a further test, cells were exposed to BSO/DEM in order to deplete GSH and then exposed to menadione. The synthesis of hsc70 following exposure to menadione was greatly increased in GSH-depleted cells compared with GSH-replete cells. Experiments were conducted to determine if electroporation of cells in GSSG containing buffer affected hsp synthesis. Electroporation in glucose-free buffer containing 3 mM GSSG did not affect hsp synthesis. We interpret these results to indicate that the inability to maintain glutathione in a reduced form during menadione redox cycling resulted in protein thiol oxidation. This, in turn, resulted in accumulation of hsc70 mRNA with a subsequent increase in the synthesis of hsc70.

The question of whether depletion of glutathione (GSH) could affect the synthesis of stress proteins was investigated in Hep G2 cells. Cells were exposed to BSO/DEM at 37 degrees C to deplete glutathione. When 95% of the glutathione was depleted cells were washed, and BSO was added to cells previously exposed to BSO/DEM; then the cells were incubated at 37, 38.5, or 39 degrees C for 4 h. Two-dimensional PAGE analysis of GSH-depleted cells incubated at 37 degrees C indicated increased synthesis of heme oxygenase and a polypeptide tentatively identified as hsp-70B'. Depletion of GSH did not affect the cellular concentration of hsp-70 as assessed by Western immunoblotting, yet Northern blot analysis indicated that hsp-70 mRNA was increased in GSH-depleted cells. Incubation of GSH-replete cells at 38.5 degrees C did not appear to enhance the amount of hsp-70 mRNA or the relative rate of hsp-70 synthesis. In contrast, incubation of GSH-depleted cells at 38.5 degrees C elevated steady-state hsp-70 mRNA levels and the rate of hsp-70 synthesis relative to total protein synthesis. Depletion of GSH also increased the relative rate of hsp-70 synthesis at 39 degrees C. These results suggest that the synthesis of stress proteins can be affected by glutathione concentrations.

The concentration of glutathione (GSH) and the expressions of gamma-glutamylcysteine 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 irradiated, and a normal-immortalized cell line (X-REF-23) was isolated. Chronic exposure of X-REF-23 cells to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) yielded cells (X-REF-23-TP) capable of benign tumor formation in nude mice. These cells exhibited GSH concentrations and gamma-glutamylcysteine synthetase heavy subunit mRNA levels that were approximately 50% less than those measured in X-REF-23 cells. Neither X-REF-23 nor X-REF-23-TP cells exhibited detectable GGT mRNA or activity. Administration of 3 Gy of X-rays followed by chronic TPA treatment yielded cells (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-glutamylcysteine synthetase heavy subunit mRNA levels were similar to those of X-REF-TP cells. X-REF-23-TPX.1 cells contained nearly the same amount 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 with X-REF-23 cells. Furthermore, exposure of X-REF-23-TPX.1 cells to DEM stimulated GSH resynthesis such that the GSH concentration exceeded control values during exposure. The resynthesis of GSH during a DEM exposure was found to be dependent upon the expression of GGT, as demonstrated by inhibition with AT-125. These experiments indicate that ionizing radiation can lead to elevated constitutive expression of GGT in transformed REF cells and that expression of GGT activity was responsible for the increased rate of GSH repletion observed in X-REF-23-TPX.1 cells.