RKO36 cells, a subclone of RKO
colorectal carcinoma cells that have been stably transfected with the pCMV-EGFP2Xho vector, were grown to confluence and then exposed to either the radioprotector
WR-1065, i.e. the active
thiol form of
amifostine, for 30 min at doses of 40 microM and 4 mM or the
cytokine tumor necrosis factor alpha (
TNFalpha, TNFA) for 30 min at a concentration of 10 ng/ml and then washed. Total
protein was isolated as a function of time up to 32 h after these treatments. Both doses of
WR-1065 as well as the concentration of
TNFalpha used were effective in elevating intracellular levels of the
antioxidant protein SOD2 (also known as MnSOD) at least 15-fold over background levels as determined by Western blot analysis, while measured SOD2 activity was elevated between 5.5- and 6.9-fold. SOD2 reached a maximal level 24 h and 20 h after
WR-1065 and
TNFalpha treatments, respectively. The
antioxidant proteins catalase and
glutathione peroxidase (GPX) were also monitored over the 32-h period. In contrast to the robust changes observed in intracellular levels of SOD2 as a function of time after exposure of cells to
WR-1065,
catalase levels were elevated only 2.6-fold over background as determined by Western blot analysis, while GPX activity was unaffected by
WR-1065 exposure. GPX
protein levels were extremely low in cells, and analysis of GPX activity using a spectrophotometric method based on the consumption of reduced
NADPH also revealed no measurable change as a function of
WR-1065 or
TNFalpha exposure. RKO36 cells either were irradiated with X rays in the presence of either 40 microM or 4 mM
WR-1065 or 10 ng/ml
TNFalpha or were irradiated 24 or 20 h later, respectively, when
SOD2 protein levels were most elevated. The concentrations and exposure conditions used for
WR-1065 and
TNFalpha were not cytotoxic and had no effect on plating efficiencies or cell survival compared to untreated controls. No protection or sensitization was observed for cells irradiated in the presence of 40 microM
WR-1065 or
TNFalpha. Survival was elevated 1.90-fold for cells irradiated in the presence of 4 mM
WR-1065. When RKO36 cells were irradiated with 2 Gy 24 h after 40 microM or 4 mM
WR-1065 and 20 h after
TNFalpha treatments when SOD2 levels were the most increased, survival was elevated 1.42-, 1.48- and 1.36-fold, respectively. This increased survival represents a SOD2-mediated delayed radioprotective effect. SOD2 appears to be an important
antioxidant gene whose inducible expression is an important
element in adaptive cellular responses in general, and the delayed radioprotective effect in particular. It can be induced by a range of agents including cytoprotective nonprotein
thiols such as
WR-1065 and pleiotropic
cytokines such as
TNFalpha.