1-12-dimethylspermine and 1-methylspermidine

The polyamines putrescine, spermidine and spermine are intimately involved in the regulation of cellular growth and viability. Transduction of human embryonic kidney (HEK) 293T cells with an adenovirus encoding a key polyamine catabolic enzyme, spermidine N¹-acetyltransferase 1 (SSAT1)/SAT1 (AdSAT1), leads to a rapid depletion of spermidine and spermine, arrest in cell growth and a decline in cell viability. Annexin V/propidium iodide FACS analyses, terminal uridine nucleotide end-labelling (TUNEL) and caspase 3 assays showed a clear indication of apoptosis in AdSAT1-transduced cells (at 24-72 h), but not in cells transduced with GFP-encoding adenovirus (AdGFP). Apoptosis in the polyamine-depleted cells occurs by the mitochondrial intrinsic pathway, as evidenced by loss of mitochondrial membrane potential, increase in pro-apoptotic Bax, decrease in anti-apoptotic Bcl-xl, Bcl2 and Mcl-1 and release of cytochrome c from mitochondria, upon transduction with AdSAT1. Moreover, TEM images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including cell shrinkage, nuclear fragmentation, mitochondrial alteration, vacuolization and membrane blebbing. The apoptosis appears to result largely from depletion of the polyamines spermidine and spermine, as the polyamine analogues α-methylspermidine (α-MeSpd) and N¹,N¹²-dimethylspermine (Me₂Spm) that are not substrates for SAT1 could partially restore growth and prevent apoptosis of AdSAT1-transduced cells. Inhibition of polyamine oxidases did not restore the growth of AdSAT1-transduced cells or block apoptosis, suggesting that the growth arrest and apoptosis were not induced by oxidative stress resulting from accelerated polyamine catabolism. Taken together, these data provide strong evidence that the depletion of the polyamines spermidine and spermine leads to mitochondria-mediated apoptosis.

Topics: Acetyltransferases; Antimetabolites; Apoptosis; Apoptosis Regulatory Proteins; Cell Proliferation; Cell Shape; Cell Size; HEK293 Cells; Humans; In Situ Nick-End Labeling; Kidney; Kinetics; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Recombinant Proteins; Spermidine; Spermine

Efficient syntheses of metabolically stable alpha-methylspermidine 1, alpha-methylspermine 2, and bis-alpha,alpha'-methylated spermine 3 starting from ethyl 3-aminobutyrate are described. The biological tolerance for these compounds was tested in wild-type mice and transgenic mice carrying the metallothionein promoter-driven spermidine/spermine N(1)-acetyltransferase gene (MT-SSAT). The efficient substitution of natural polyamines by their derivatives was confirmed in vivo with the rats harboring the same MT-SSAT transgene and in vitro with the immortalized fibroblasts derived from these animals. Enantiomers of previously unknown 1-amino-8-acetamido-5-azanonane dihydrochloride 4 were synthesized starting from enantiomerically pure (R)- and (S)-alaninols. The studies with recombinant human polyamine oxidase (PAO) showed that PAO (usually splits achiral substrates) strongly favors the (R)-isomer of 4 that demonstrates for the first time that the enzyme has hidden potency for stereospecificity.

Topics: Animals; Animals, Genetically Modified; Dose-Response Relationship, Drug; Drug Tolerance; Fibroblasts; Humans; In Vitro Techniques; Kidney; Liver; Mice; Molecular Structure; Oxidoreductases Acting on CH-NH Group Donors; Pancreas; Polyamine Oxidase; Rats; Recombinant Proteins; Spermidine; Spermine; Stereoisomerism; Structure-Activity Relationship; Substrate Specificity

The abilities of the natural polyamines, spermidine and spermine, and of the synthetic analogues, 1-methylspermidine and 1,12-dimethylspermine, to reverse the effects of the S-adenosyl-L-methionine decarboxylase inhibitor 5'-([(Z)-4-aminobut-2-enyl]methylamino)-5'-deoxyadenosine (AbeAdo) on L1210-cell growth were studied. L1210 cells were exposed to AbeAdo for 12 days to induce cytostasis and then exposed to spermidine, spermine, 1-methylspermidine or 1,12-dimethylspermine in the continued presence of AbeAdo. AbeAdo-induced cytostasis was overcome by the natural polyamines, spermidine and spermine. The cytostasis was also reversed by 1-methylspermidine. 1,12-Dimethylspermine had no effect on the AbeAdo-induced cytostasis of chronically treated cells, although it was active in permitting growth of cells treated with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. The initial 12-day exposure to AbeAdo elevated intracellular putrescine levels, depleted intracellular spermidine and spermine, and resulted in the accumulation of unmodified eukaryotic translation initiation factor 5A (eIF-5A). Exposure of these cells to exogenous spermidine, which is the natural substrate for deoxyhypusine synthase, resulted in a decrease in the unmodified eIF-5A content. 1-Methylspermidine, which was found to be a substrate of deoxyhypusine synthase in vitro, also decreased the levels of unmodified eIF-5A in the AbeAdo-treated cells. Although spermine is not a substrate of deoxyhypusine synthase, spermine was converted into spermidine in the L1210 cells, and spermine addition to AbeAdo-treated cells resulted in the appearance of both intracellular spermine and spermidine and in the decrease in unmodified eIF-5A. Exogenous 1,12-dimethylspermine, which was not metabolized to spermine or to 1-methylspermidine and was not a substrate of deoxyhypusine synthase in vitro, did not decrease levels of unmodified eIF-5A. The finding that AbeAdo-induced cytostasis was only reversed by polyamines and polyamine analogues that result in the formation of hypusine or an analogue in eIF-5A is consistent with the hypothesis [Byers, Wiest, Wechter and Pegg (1993) Biochem. J. 290, 115-121] that AbeAdo-induced cytostasis is due to the depletion of the hypusine-containing form of eIF-5A, which is secondary to the depletion of spermidine by inhibition of S-adenosyl-L-methionine decarboxylase.

Topics: Adenosylmethionine Decarboxylase; Animals; Cell Division; CHO Cells; Chromatography, High Pressure Liquid; Cricetinae; Deoxyadenosines; Eflornithine; Eukaryotic Translation Initiation Factor 5A; Leukemia L1210; Lysine; Oxidoreductases Acting on CH-NH Group Donors; Peptide Initiation Factors; RNA-Binding Proteins; Spermidine; Spermine; Substrate Specificity; Tumor Cells, Cultured

In order to assess the tolerance of the target enzyme spermine synthase for alpha-substituents on the aminopropyl moiety of the substrate spermidine, 1-methylspermidine (MeSpd, 2) was synthesized. It was determined that MeSpd is a poor substrate for spermine synthase and is not a substrate for spermidine N1-acetyltransferase, suggesting that alpha-methylated polyamines might be metabolically stable and therefore useful tools for studying polyamine effects in intact cells. On the basis of initial cellular results with 2, 1-methylspermine (MeSpm, 3) and 1,12-dimethylspermine (Me2Spm, 4) were also synthesized. When added to cells (L1210, SV-3T3, or HT29) depleted of both putrescine and spermidine by prior treatment with alpha-(difluoromethyl)ornithine (DFMO), these alpha-methylated polyamines were able to restore cell growth to that observed in the absence of DFMO. In accord with the enzyme data noted above, metabolic studies indicated a slow conversion of 2 to 3, but no metabolism of 4 in these cells. It was concluded from these results that the alpha-methylated polyamines are able to substitute for the natural polyamines spermidine and spermine in critical biochemical processes which involve polyamines for continued cell growth. In accord with the hypothesis, preliminary data indicate that MeSpd and Me2Spm are as effective as spermidine and spermine, respectively, in promoting the conversion of B-DNA to Z-DNA.

Topics: Animals; Cell Division; Cell Line, Transformed; Colonic Neoplasms; Eflornithine; Humans; Leukemia L1210; Methylation; Mice; Putrescine; Spermidine; Spermine; Spermine Synthase; Substrate Specificity; Tumor Cells, Cultured