The polyamines putrescine, spermidine and spermine are organic cations shown to participate in a bewildering number of cellular reactions, yet their exact functions in intermediary metabolism and specific interactions with cellular components remain largely elusive. Pharmacological interventions have demonstrated convincingly that a steady supply of these compounds is a prerequisite for cell proliferation to occur. The last decade has witnessed the appearance of a substantial number of studies, in which genetic engineering of polyamine metabolism in transgenic rodents has been employed to unravel their cellular functions. Transgenic activation of polyamine biosynthesis through an overexpression of their biosynthetic enzymes has assigned specific roles for these compounds in spermatogenesis, skin physiology, promotion of tumorigenesis and organ hypertrophy as well as neuronal protection. Transgenic activation of polyamine catabolism not only profoundly disturbs polyamine homeostasis in most tissues, but also creates a complex phenotype affecting skin, female fertility, fat depots, pancreatic integrity and regenerative growth. Transgenic expression of ornithine decarboxylase antizyme has suggested that this unique protein may act as a general tumor suppressor. Homozygous deficiency of the key biosynthetic enzymes of the polyamines, ornithine and S-adenosylmethionine decarboxylase, as achieved through targeted disruption of their genes, is not compatible with murine embryogenesis. Finally, the first reports of human diseases apparently caused by mutations or rearrangements of the genes involved in polyamine metabolism have appeared.Keywords: antizyme; ornithine decarboxylase; putrescine; spermidine/spermine N 1 -acetyltransferase; spermidine; spermine; transgenic mouse; transgenic rat. IntroductionThe cellular functions of the natural polyamines (putrescine, spermidine and spermine) are still largely unknown, although a vast number of studies have shown that these polycationic compounds are crucial to the growth and proliferation of mammalian cells. Pharmacological approaches are applied typically in studies aimed to unravel their functions in cellular metabolism and, admittedly, much valuable information has been generated with the use of specific inhibitors of polyamine biosynthesis. However, the last decade has produced a substantial number of experimental studies in which genetic engineering of polyamine metabolism has been used as a tool to elucidate their cellular functions. Studies with genetically engineered mice and rats have not only brought entirely new information about the involvement of polyamines in various physiological and pathophysiological processes but they have likewise challenged some of the conventional wisdoms. Mainly, four different approaches have been applied in the genetic engineering of experimental animals: (a) activation of polyamine biosynthesis through the overexpression of their biosynthetic enzymes; (b) activation of polyamine catabolism through the overexpression of the enzymes i...

Polyamines are required for optimal growth and function of cells. Regulation of their cellular homeostasis is therefore tightly controlled. The key regulatory enzyme for polyamine catabolism is the spermidine͞spermine N 1 -acetyltransferase (SSAT). Depletion of cellular polyamines has been associated with inhibition of growth and programmed cell death. To investigate the physiological function SSAT, we generated a transgenic rat line overexpressing the SSAT gene under the control of the inducible mouse metallothionein I promoter. Administration of zinc resulted in a marked induction of pancreatic SSAT, overaccumulation of putrescine, and appearance of N 1 -acetylspermidine with extensive depletion of spermidine and spermine in transgenic animals. The activation of pancreatic polyamine catabolism resulted in acute pancreatitis. In nontransgenic animals, an equal dose of zinc did not affect pancreatic polyamine pools, nor did it induce pancreatitis. Acetylated polyamines, products of the SSAT-catalyzed reaction, are metabolized further by the polyamine oxidase (PAO) generating hydrogen peroxide, which might cause or contribute to the pancreatic inflammatory process. Administration of specific PAO inhibitor, MDL72527 [N 1 ,N 2 -bis(2,3-butadienyl)-1,4-butanediamine], however, did not affect the histological score of the pancreatitis. Induction of SSAT by the polyamine analogue N 1 ,N 11 -diethylnorspermine reduced pancreatic polyamines levels only moderately and without signs of organ inflammation. In contrast, the combination of N 1 ,N 11 -diethylnorspermine with MDL72527 dramatically activated SSAT, causing profound depletion of pancreatic polyamines and acute pancreatitis. These results demonstrate that acute induction of SSAT leads to pancreatic inflammation, suggesting that sufficient pools of higher polyamine levels are essential to maintain pancreatic integrity. This inflammatory process is independent of the production of hydrogen peroxide by PAO.I ntracellular polyamine levels are tightly maintained by a number of mechanisms, suggesting their importance for cell function. This polyamine homeostasis is controlled by their biosynthesis, interconversion, catabolism, secretion, and uptake. Polyamine biosynthesis is regulated by the activities of ornithine and S-adenosylmethionine decarboxylases. Overexpression of ornithine decarboxylase in transgenic rodents affected spermatogenesis (1, 2), rendered animals more resistant to chemically or electrically induced seizure activity (3) and to ischemic insults (4), and enhanced skin papilloma formation (5). However, the activation of polyamine biosynthesis caused by overexpression of ornithine decarboxylase did not result in enhanced accumulation of the higher polyamines spermidine and spermine. Polyamine catabolism is controlled by the activity of spermidine͞spermine N 1 -acetyltransferase (SSAT; refs. 6 and 7). The acetylated products are oxidized further by the polyamine oxidase (PAO) to spermidine and putrescine. We recently generated transgenic mouse lines with ...

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We have earlier shown that ␣-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because ␣-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because ␣,-bismethylspermine can be converted to methylspermidine, it is not entirely clear whether the restoration of cell growth is actually attributable to hypusine formed from these polyamine analogues. Here, we have used optically active isomers of methylated spermidine and spermine and show that polyamine depletion-induced acute cytostasis in cultured cells could be reversed by all the isomers of the methylpolyamines irrespective of whether they served or not as precursors of hypusine. In transgenic rats with activated polyamine catabolism, all the isomers similarly restored liver regeneration and reduced plasma ␣-amylase activity associated with induced pancreatitis. Under the above experimental conditions, the (S,S)-but not the (R,R)-isomer of bismethylspermine was converted to methylspermidine apparently through the action of spermine oxidase strongly preferring the (S,S)-isomer. Of the analogues, however, only (S)-methylspermidine sustained cell growth during prolonged (more than 1 week) inhibition of polyamine biosynthesis. It was also the only isomer efficiently converted to hypusine, indicating that deoxyhypusine synthase likewise possesses hidden stereospecificity. Taken together, the results show that growth inhibition in response to polyamine depletion involves two phases, an acute and a late hypusine-dependent phase.A large number of studies have indicated that a continuous supply of the polyamines (spermidine and spermine) is required for animal cell proliferation to occur as polyamine depletion resulted either from a specific inhibition of their biosynthesis (1) or from an activation of their catabolism (2) invariably leads to growth inhibition. The molecular mechanisms involved in the requirement of polyamines for animal cell growth are largely unknown besides the fact that spermidine, but not spermine, serves as the sole biosynthetic precursor for hypusine, an unusual amino acid that is an integral component of eukaryotic translation initiation factor 5A (eIF5A) 4 (3). Because functional (hypusinated) eIF5A is required for animal cell growth (4, 5), it is often difficult to judge whether spermidine depletion-induced growth inhibition is secondary to hypusine deprivation. The finding that cytostasis resulting from an inhibition of S-adenosylmethionine decarboxylase in cultured cells is reversed by ␣-methylspermidine (MeSpd) but not by ␣,-bismethylspermine (Me 2 Spm) indicates that growth inhibition was attributable to hypusine depletion as MeSpd, but not Me 2 Spm, can serve as the biosynthetic precursor for hypusine formation (6). On the other hand, MeSpd and both singly and doubly methylated spermine deri...

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