Inhibition of NFκB activation by dominant-negative IκBα abrogates the delayed radioprotective effect induced by amifostine’s active metabolite WR1065.

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Delayed radioprotection, the enhanced radiation resistance of cells 24 h after their exposure to thiol-containing drugs such as WR1065, correlates with the time interval when intracellular levels of Sod2 are maximally elevated. The proposed underlying mechanism of action for this effect involves the activation of NFκB that leads to enhanced expression of Sod2. We have further characterized the effects of WR1065 on NFκB and Sod2 enzyme levels as they relate to the delayed radioprotective effect. Using mobility shift assays, the ability of WR1065 to activate NFκB in SA-NH cells stably transfected with dominant-negative mutant IκBα (designated SA-NH+mIκBα1) was compared with non-transfected SA-NH cells. Treatment of cells with 4 mM WR1065 for 30 min induced activation of NFκB in SA-NH cells, but not in SA-NH+mIκBα1 cells. To assess the effects of WR1065 exposure on Sod2 levels, SA-NH+mIκBα1 cells were treated with either 40 μM (anti-mutagenic dose) or 4 mM (cytoprotective dose) WR1065 for 30 min, washed free of the drug, and cultured in drug-free medium for 24 h. At that time, relative Sod2 protein levels were determined by Western blotting. No increase in Sod2 levels in drug treated cells was observed relative to untreated control cells, in contrast to a 10-fold enhancement we previously observed in SA-NH cells 24 h after WR1065 exposure. Growth characteristics and clonogenic survival following exposure to x-rays were examined in both SA-NH and SA-NH+mIκBα1 cells. As determined by flow cytometry, no significant difference in the cell cycle patterns was observed for either cell line under both log and confluent phases of growth. Compared to parental SA-NH cells, SA-NH+mIκBα1 cells are slightly more radiosensitive (α = 0.278 ± 0.040 [SEM], β = 0.096 ± 0.007, α/β = 2.893 ± 0.595 versus α = 0.150 ± 0.021, β = 0.044 ± 0.003, α/β = 3.409 ± 0.723). Clonogenic assays were also performed to determine if WR1065 could protect SA-NH+mIκBα1 cells against radiation-induced toxicity. Cells were treated with either 40 μM or 4 mM WR1065 for 30 min and irradiated with x-rays in the presence of the drug or with the two concentrations of the drug for 30 min, washed, and incubated in drug-free medium for 24 h before being irradiated. Under the first experimental condition, protection against cell killing was observed for those cells pretreated with 4 mM WR1065 and irradiated in the presence of the drug. In contrast, no cytoprotection was observed for SA-NH+mIκBα1 cells irradiated 24 h after exposure to either 40 μM or 4 mM WR1065, the time frame during which the maximum elevation in Sod2 levels is observed in the parental SA-NH cells. These data support our hypothesis that the delayed cytoprotection observed in cells 24 h following exposure to WR1065 is the result of its ability to induce NFκB activity and enhance Sod2 enzyme levels. This work was supported by NIH/NCI grants RO1-CA37435 and CA99005 (D.J.G.).