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N1,N11-diethylnorspermine (DENSPM) is a polyamine analog that down-regulates polyamine biosynthesis and potently upregulates the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). In certain cells, such as SKMEL-28 human melanoma cells, induction of SSAT is associated with rapid apoptosis. In this study, we used small interfering RNA (siRNA) to examine the role of SSAT induction in mediating polyamine pool depletion and apoptosis. siRNA duplexes were designed to target three independent sites in the SSAT mRNA coding region (siSSAT). When transfected under nontoxic conditions, two of the duplexes selectively reduced basal SSAT mRNA in HEK-293 cells by >80% and prevented DENSPM-induced SSAT mRNA by 95% in SK-MEL-28 cells. Treatment of SK-MEL-28 cells with 10 muM DENSPM in the presence of 83 nM siSSAT selectively prevented the 1400-fold induction of SSAT activity by approximately 90% and, in turn, prevented analog depletion of spermine (Spm) pools by approximately 35%. siSSAT also prevented DENSPM-induced cytochrome c release and caspase-3 cleavage at 36 h and apoptosis at 48 h as measured by annexin V staining. Overall, the data directly link analog induction of SSAT to Spm pool depletion and to caspase-dependent apoptosis in DENSPM-treated SK-MEL-28 cells. This represents the first use of siRNA technology directed toward a polyamine gene and the first unequivocal demonstration that SSAT induction initiates events leading to polyamine analog-induced apoptosis.

We have recently generated transgenic mice in which polyamine catabolism has been activated by overexpressing the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT). These animals have now been tested for their sensitivity to the polyamine analog N1,N11-diethylnorspermine (DENSPM), which is currently undergoing Phase I clinical trial. The analog is known for its ability to potently induce SSAT. Treatment for 4 days with a daily dose (125 mg/kg) of analog caused profound changes in polyamine metabolism in the transgenic animals. Liver SSAT activity was increased by approximately 800-fold while hepatic mRNA increased only 4-fold. Putrescine pools increased while spermidine and spermine pools nearly disappeared, resulting in a compensatory increase in ornithine decarboxylase activity. Similar but less profound changes were also seen in other tissues (spleen, intestine, and skin). This treatment also resulted in a 50% mortality in the transgenic animals, with no apparent histopathological changes in major organs. Nontransgenic animals exhibited no toxicity, and tissue SSAT activity was unchanged or only moderately increased. Polyamine pools were only slightly altered. Greater analog toxicity in transgenic animals may be attributable to higher tissue levels of DENSPM facilitated by SSAT-mediated decreases in spermidine and spermine. To further confirm the enhanced sensitivity of the transgenic animals to the analog, groups of nontransgenic and transgenic animals were subjected to daily injections with DENSPM. On average, transgenic mice died approximately 3 days earlier than their nontransgenic litter-mates. The findings indicate a contributing role for SSAT in whole animal toxicity by SSAT-inducing polyamine analogs.

The spermine analog N1,N11-diethylnorspermine (DE-333, also known as DENSPM or BENSPM) is regarded as the most potent known inducer of the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT), increasing activity by more than 200- to 1000-fold in certain cell types. The relative ability of a series of eight systematically modified DE-333 analogs to affect SSAT expression was examined in Malme-3M human melanoma cells, one of several cell lines known to be especially responsive to induction of this enzyme. In particular, we examined the relative contribution of induction of enzyme mRNA and prolongation of enzyme half-life to analog-mediated increases in enzyme activity. Induction of enzyme mRNA was most influenced by intra-amine carbon distances; relative effectiveness was found to be proportional to the number of three-carbon units. Stabilization of enzyme was most determined by the terminal N-alkyl substituent size; among methyl, ethyl and propyl groups, methyl was least effective. Thus, DE-333, which most potently induces SSAT mRNA and effectively stabilizes SSAT enzyme activity, produces the greatest increase in enzyme activity. Although other contributing mechanisms may be involved, the relative abilities of the various analogs to induce enzyme activity is at least partially attributable to their combined effects on enzyme mRNA and protein half-life. These data reveal the highly sensitive structure-activity relationships that underlie and control spermine analog induction of SSAT activity. Pending further definition of the relationship between SSAT induction and antitumor growth and toxicity in vivo, these relationships may be used to optimize therapeutic efficacy.