Abstract
Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, we reasoned that this might be more effectively achieved by activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT); a strategy first validated in MCF-7 breast carcinoma cells. We now examine the possibility that, due to unique aspects of polyamine homeostasis in the prostate gland, tumor cells derived from it may be particularly sensitive to activated polyamine catabolism. Thus, SSAT was conditionally overexpressed in LNCaP prostate carcinoma cells via a tetracycline-regulatable (Tet-off) system. Tetracycline removal resulted in a rapid approximately 10-fold increase in SSAT mRNA and an increase of approximately 20-fold in enzyme activity. SSAT products N(1)-acetylspermidine, N(1)-acetylspermine, and N(1),N(12)-diacetylspermine accumulated intracellularly and extracellularly. SSAT induction also led to a growth inhibition that was not accompanied by polyamine pool depletion as it was in MCF-7 cells. Rather, intracellular spermidine and spermine pools were maintained at or above control levels by a robust compensatory increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase activities. This, in turn, gave rise to a high rate of metabolic flux through both the biosynthetic and catabolic arms of polyamine metabolism. Treatment with the biosynthesis inhibitor alpha-difluoromethylornithine during tetracycline removal interrupted flux and prevented growth inhibition. Thus, flux-induced growth inhibition appears to derive from overaccumulation of metabolic products and/or from depletion of metabolic precursors. Metabolic effects that were not excluded as possible contributing factors include high levels of putrescine and acetylated polyamines, a 50% reduction in S-adenosylmethionine, and a 45% decline in the SSAT cofactor acetyl-CoA. Overall, the study demonstrates that activation of polyamine catabolism in LNCaP cells elicits a compensatory increase in polyamine biosynthesis and downstream metabolic events that culminate in growth inhibition.
Highlights
Of biosynthesis, catabolism, uptake, and export, each of which is sensitively regulated by effector molecules that, in turn, are controlled by intracellular polyamine pools [1]
As an alternative approach to the use of enzyme inhibition, we propose that disruption of polyamine homeostasis at the level of polyamine catabolism may have unique therapeutic potential against prostate carcinoma
In an earlier report [31], we showed that conditional overexpression of spermidine/spermine N1-acetyltransferase (SSAT) leads to polyamine pool depletion and growth inhibition in MCF-7 breast carcinoma cells
Summary
Materials—The inhibitor of polyamine oxidase (PAO), N1-methyl-N2(2,3-butadienyl)butane-1,4-diamine (MDL-72527) was generously provided by Aventis Pharmaceuticals Inc. (Bridgewater, NJ). Intracellular polyamines, including acetylated derivatives of spermidine (Spd) and Spm were extracted from cell pellets with 0.6 N perchloric acid, dansylated, measured by reverse phase high-performance liquid chromatography (HPLC) as described by Kramer et al [38], and expressed as picomoles/106 cells. Extracellular polyamines and acetylated polyamines were extracted from media as described by Kramer et al [39], containing fetal bovine serum, Tet, and L-glutamine but not G418 or hygromycin B. S-Adenosylmethionine and Metabolite Pools—Intracellular SAM and its metabolites, dcSAM and MTA, were extracted from cell pellets with 0.6 N perchloric acid and measured by HPLC according to chromatographic conditions reported by Yarlett and Bacchi [40] with modifications as described by Kramer et al [38]. Effluent was monitored with a Waters 2487 dual wavelength UV detector, and data were processed using instrumentation described for polyamine pool analysis and expressed as picomoles/106 cells.
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