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Regulation of S-adenosylmethionine decarboxylase activity by alterations in the intracellular polyamine content.

ABSTRACT: The effects of addition of exogenous spermidine and spermine and of two inhibitors of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), which decreases spermidine concentrations, and n-butyl-1,3-diaminopropane, which depletes spermine, on the expression of S-adenosylmethionine decarboxylase (AdoMetDC) activity were studied in mammalian cell lines (HT29, CHO and COS-7). AdoMetDC levels were inversely related to the polyamine content, and spermine was the more potent repressor of AdoMetDC activity, but only spermidine affected the amount of AdoMetDC mRNA. Transfection of COS-7 cells or CHO cells with plasmid constructs containing a chloramphenicol acetyltransferase (CAT) reporter gene driven by portions of the AdoMetDC promoter region indicated that CAT expression was altered by spermidine, but not by spermine, suggesting that there is a spermidine-responsive element in this promoter. Transient transfection of COS-7 cells with pSAMh1, a plasmid containing the AdoMetDC cDNA in a vector with the SV40 promoter and origin of replication, led to a large increase in AdoMetDC expression. Although treatment of COS-7 cells with n-butyl-1,3-diaminopropane greatly increased endogenous AdoMetDC activity, the spermine depletion brought about by this inhibitor did not stimulate AdoMetDC expression from pSAMh1. The pSAMh1 cDNA is missing 72 nucleotides from the 5' end of the AdoMetDC mRNA, and it is possible that translational regulation by spermine involves this region. The expression of AdoMetDC from pSAMh1 in COS-7 cells was greatly inhibited by DFMO treatment, although endogenous AdoMetDC activity was increased. The expression of other plasmids containing the SV40 origin of replication was also inhibited by DFMO in COS-7 cells, but not in CHO cells. DFMO treatment did not interfere with the expression of plasmids driven by the RSV promoter. These results suggest that low spermidine levels interfere with the replication of plasmids containing the SV40 origin of replication.

SUBMITTER: Shantz LM

PROVIDER: S-EPMC1132040 | biostudies-other | 1992 Dec

REPOSITORIES: biostudies-other

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Project description:1. The content of decarboxylated S-adenosylmethionine (AdoMet) in transformed mouse fibroblasts (SV-3T3 cells) was increased 500-fold to about 0.4fmol/cell when ornithine decarboxylase was inhibited by alpha-difluoromethylornithine. This increase was due to the absence of putrescine and spermidine, which serve as substrates for aminopropyltransferases with decarboxylated AdoMet as an aminopropyl donor, and to the enhanced activity of AdoMet decarboxylase brought about by depletion of spermidine. The increase in decarboxylated AdoMet content was abolished by addition of putrescine, but not by 1,3-diaminopropane. 2. 5'-Methylthiotubercidin also increased decarboxylated AdoMet content, presumably by direct inhibition of aminopropyl-transferase activities, but the increase in its content and the decline in spermidine content were much less than those produced by alpha-difluoromethylornithine. 3. Decarboxylated AdoMet content of regenerating rat liver was measured in rats treated with inhibitors of ornithine decarboxylase. The content was increased by 60% 32h after partial hepatectomy in control rats, by 90% when alpha-difluoromethylornithine was given to the partially hepatectomized rats, and by 330% when 1,3-diaminopropane was used to inhibit putrescine and spermidine synthesis. After 48h of exposure to 1,3-diaminopropane, which completely prevented the increase in spermidine after partial hepatectomy, there was a 5-fold rise in hepatic decarboxylated AdoMet concentration. These increases were prevented by treatment with putrescine or with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase. 4. These results show that changes in AdoMet metabolism result from the administration of specific inhibitors of polyamine synthesis. The possible consequences of the accumulation of decarboxylated AdoMet, which could, for example, interfere with normal cellular methylation or lead to depletion of cellular adenine nucleotides, should be considered in the interpretation of results obtained with such inhibitors.

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Regulation of S-adenosylmethionine decarboxylase activity by alterations in the intracellular polyamine content.

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