Spermidine/spermine N(1)-acetyltransferase (SSAT) is a key enzyme in polyamine catabolism. We recently reported that the combination of N(1), N(11)-diethylnorspermine (DENSPM) and 5-fluorouracil (5-FU) synergistically induces SSAT expression, depletes polyamine levels and causes apoptosis in colon cancer cells. To determine whether new RNA and protein synthesis is required for SSAT induction, we examined the effect of actinomycin D (ActD) and cycloheximide (CHX). ActD alone blocked the induction of SSAT expression; however, the combination of CHX and DENSPM markedly induced SSAT expression and caused mitochondrial damage, suggesting that an inhibitory labile protein is involved in SSAT transactivation. SSAT promoter analysis identified two putative Rel/Nuclear Factor kappaB (NFkappaB) binding sites. Thus, we hypothesized that IkappaB is the labile inhibitory protein and that its removal contributes to the activation of NFkappaB. CHX quickly eliminated the IkappaB protein in the cells and increased the levels of the two subunits of NFkappaB, p65 and p50, in the nucleus. Luciferase reporter gene assay showed that SSAT promoter constructs containing the two putative NFkappaB binding elements responded to CHX as well as TNFalpha, whereas the promoter without the two sites did not. Chromatin immunoprecipitation (ChIP) assay showed that NFkappaB was indeed bound to the SSAT promoter after CHX treatment. Further, dominant negative IkappaB attenuated the CHX and DENSPM-induced SSAT expression and mitochondria damage. These results taken together suggest that the inhibition of IkappaB and activation of NFkappaB activate SSAT.