Correlation of Polyamine and Growth Responses to N1,N11-Diethylnorspermine in Primary Fetal Fibroblasts Derived from Transgenic Mice Overexpressing Spermidine/SpermineN1-Acetyltransferase*
Abstract
A recently generated transgenic mouse line having activated polyamine catabolism due to systemic overexpression of spermidine/spermineN 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 ∼20- and ∼40-fold higher than in nontransgenic fibroblasts. Consistent with activated polyamine catabolism, there was an overaccumulation of putrescine andN 1-acetylspermidine and a decrease in spermidine and spermine pools. Treatment with the polyamine analogueN 1 ,N 11-diethylnorspermine (DENSPM) increased SSAT activity in the transgenic fibroblasts ∼380-fold, whereas mRNA increased only ∼3-fold, indicating post-mRNA regulation. SSAT activity in the nontransgenic fibroblasts increased ∼200-fold. By Western blot, enzyme protein was found to increase ∼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.
Footnotes
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↵* This work was supported in part by Grants CA-65942 and CA-22153 from the National Cancer Institute, National Institutes of Health, by the Academy of Finland, and by Human Frontier Science Program, and by a International Union against Cancer fellowship (to J. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence and reprint requests should be addressed: Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Sts., Buffalo, NY 14263. Tel.: 716-845-3002; Fax: 716-845-8857; E-mail: porter{at}sc3101.med.buffalo.edu.
- Received August 14, 1997.
- Revision received October 8, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
