Biaglow, J. E., Ayene, I. S., Koch, C. J., Donahue, J., Stamato, T. D., Mieyal, J. J. and Tuttle, S. W. Radiation Response of Cells during Altered Protein Thiol Redox. Radiat. Res. 159, 484–494 (2003).
The major focus of this work was to investigate how altered protein thiol redox homeostasis affects radiation-induced cell death. We used the cells of wild-type CHO cell line K1, the CHO cell line E89, which is null for G6PD activity, and a radiation-sensitive CHO cell line, XRS5. The protein-thiol redox status of cells was altered with cell-permeable disulfides, hydroxyethyldisulfide (HEDS) or lipoate. HEDS is primarily reduced by thioltransferase (glutaredoxin), with GSH as the electron donor. In contrast, lipoate is reduced by thioredoxin reductase. HEDS was reduced at a greater rate than lipoate by G6PD-containing K1 (wild-type) cells. Reduction of disulfides by G6PD-deficient cells was significantly slower with HEDS as substrate and was nearly absent with lipoate. The rate of reduction of HEDS by E89 cells decelerated to near zero by 30 min, whereas the reduction continued at nearly the same rate during the entire measurement period for K1 cells. HEDS treatment decreased the GSH and protein thiol (PSH) content more in G6PD-deficient cells than in G6PD-containing cells. On the other hand, lipoate did not significantly alter the protein thiol, but it increased the GSH in K1 cells. Acute depletion of GSH by l-buthionine-sulfoximine (l-BSO) in combination with dimethylfumarate significantly decreased the rate of reduction of HEDS by K1 cells close to that of G6PD-deficient cells. Prior GSH depletion by l-BSO alone significantly decreased the PSH in glucose-depleted E89 cells exposed to HEDS, but this did not occur with K1 cells. The radiation response of G6PD-deficient cells was significantly sensitized by HEDS, but HEDS did not have this effect on K1 cells. The DNA repair-deficient XRS5 CHO cells displayed the same capacity as K1 cells for HEDS reduction, and like K1 cells the XRS5 cells were not sensitized to radiation by HEDS treatment. Deprivation of glucose, which provides the substrate for G6PD in the oxidative pentose phosphate cycle, decreased the rate of bioreduction of HEDS and lipoate in G6PD-containing cells to the level in G6PD-deficient cells. In the absence of glucose, HEDS treatment diminished non-protein thiol and protein thiol to the same level as those in G6PD-deficient cells and sensitized the K1 cells to HEDS treatment. However, depletion of glucose did not alter the sensitivity of XRS5 cells in either the presence or absence of HEDS. Overall the results suggest a major role for pentose cycle control of protein redox state coupled to the activities of the thioltransferase and thioredoxin systems. The results also show that protein thiol status is a critical factor in cell survival after irradiation.