Correlation of polyamine and growth responses to N1,N11-diethylnorspermine in primary fetal fibroblasts derived from transgenic mice overexpressing spermidine/spermine N1-acetyltransferase

A recently generated transgenic mouse line having activated polyamine catabolism due to systemic overexpression of spermidine/spermine N-1-acetyltransferase (SSAT) was used to isolate primary fetal fibroblasts as a means to further elucidate the cellular consequences of activated polyamine catabolism, Basal levels of SSAT activity and steady-state mRNA in the transgenic fibroblasts were about similar to 20- and similar to 40-fold higher than in nontransgenic fibroblasts, Consistent with activated polyamine catabolism, there was an overaccumulation of putrescine and N-1-acetylspermidine and a decrease in spermidine and spermine pools. Treatment with the polyamine analogue N-1,N-11-diethylnorspermine (DENSPM) increased SSAT activity in the transgenic fibroblasts similar to 380-fold, whereas mRNA increased only similar to 3-fold, indicating post-mRNA regulation, SSAT activity in the nontransgenic fibroblasts increased similar to 200-fold, By Western blot, enzyme protein was found to increase similar to 46 times higher in the treated transgenic fibroblasts than non-transgenic fibroblasts: a value comparable to 36-fold differential in enzyme activity, With DENSPM treatment, spermidine pools were more rapidly depleted in the transgenic fibroblasts than in nontransgenic fibroblasts, Similarly, transgenic fibroblasts were much more sensitive to DENSPM-induced growth inhibition, This was not diminished by co-treatment with an inhibitor of polyamine oxidase, suggesting that growth inhibition was due to polyamine depletion per se as opposed to oxidative stress, Since the two fibroblasts were genetically identical except for the transgene, the various metabolic and growth response differences are directly attributable to overexpression of SSAT.