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...
Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of ␣-methylspermidine, ␣-methylspermine, and bis-␣-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N 1 -acetyltransferase. ␣-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, ␣-methylspermine was readily converted to ␣-methylspermidine and spermidine; similarly, bis-␣-methylspermine was converted to ␣-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N 1 ,N 2 -bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N 1 -acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both ␣-methylspermidine and bis-␣-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.Although the requirement of the natural polyamines spermidine, spermine, and their precursor putrescine for the growth of mammalian cells is extremely well documented, their specific functions in proliferative processes are largely unknown (1). Some of the published data appear to assign a central role to spermidine, whereas putrescine is supposed to serve as its precursor and spermine as a storage pool convertible back to spermidine. For the elucidation of the physiological roles of individual polyamines, metabolically stable derivatives of polyamines fulfilling their specific cellular functions would be extremely valuable. Methyl derivatives of spermidine and spermine have been used as substitutes for the natural polyamines both in vitro and in vivo. ␣-Methylspermidine (MeSpd), 1 ␣-methylspermine (MeSpm), and bis-␣-methylspermine (1,12-dimethylspermine, Me 2 Spm) are equally effective as the natural polyamines in inducing the conversion of right-handed B-DNA to left-handed Z-DNA (2). In addition to spermidine and spermine, cytostasis that resulted from the inhibition of the S-adenosylmethionine decarboxylase can be reversed by MeSpd but not by Me 2 Spm (3). Spermidine, spermine (because of its conversion to spermidine), and MeSpd serve as substrates for the synthesis of deoxyhypusine (an integral part of eukaryotic initiation factor 5A), whereas Me 2 Spm does not (3). Interestingly, all of the mentioned methylated derivatives of spermidine and spermine have been reported to reverse the cytostasis induced by difluoromethylornithine, a specific inhibitor of mammalian ornithine decarboxyl...
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