Delayed cytoprotection after enhancement of Sod2 (MnSOD) gene expression in SA-NH mouse sarcoma cells exposed to WR-1065, the active metabolite of amifostine.

Abstract

SA-NH mouse sarcoma cells were grown to confluence and then exposed to either 40 microM or 4 mM of WR-1065, i.e. the active thiol form of amifostine, for 30 min and then washed. Total RNA and protein were isolated at various times up to 24 h after exposure. Both concentrations of WR-1065 were equally effective in affecting Sod2 (also known as MnSOD) gene expression and protein levels. Northern blot analysis using a mouse cDNA probe revealed three Sod2 transcripts of 1, 4 and 6 kb. Expression of both the 4- and 6-kb transcripts increased by 20 and 60%, respectively, and remained elevated over a period of 4 to 20 h. Sod2 protein levels, as determined by Western blot analysis, increased 15-fold over background control levels over the same interval. Sod2 protein was evaluated using activity gels and was found to be active. SA-NH cells were irradiated with X rays either in the presence of 40 microM or 4 mM WR-1065 or 24 h later after its removal, when Sod2 protein levels were most elevated. No protection was observed for cells irradiated in the presence of 40 microM WR-1065. In contrast, survival after a dose of 2 Gy was elevated 1.27-, 1.14- and 1.20-fold in SA-NH cells irradiated in the presence of 4 mM WR-1065 or 24 h after exposure of the cells to 40 microM and 4 mM WR-1065, respectively. The increased survival levels observed 24 h after exposure to WR-1065 represents a delayed radioprotective effect of WR-1065 and corresponds to the time at which Sod2 protein levels are most elevated. These data demonstrate a novel mechanism for radioprotection by WR-1065 and suggest a new potential concern regarding the issue of tumor protection.