Feedback regulation of mammalian ornithine decarboxylase. Studies using a transient expression system.

Ornithine decarboxylase catalyzes the first step in the biosynthesis of polyamines in mammalian cells. The enzyme is subject to various control mechanisms to maintain adequate intracellular levels of polyamines. Polyamines exert a strong feedback control on ornithine decarboxylase. In a recent study [van Daalen Wetters, T., Macrae, M., Brabant, M., Sittler, A. & Coffino, P. (1989) Mol. Cell. Biol. 9, 5484-5490], it was concluded that feedback control of ornithine decarboxylase is mainly, if not exclusively, a posttranslational phenomenon. The existence of a fast-acting polyamine-stimulated component of ornithine decarboxylase degradation that acts on newly synthesized monomeric forms of the enzyme was postulated. In the present study we have used a transient expression system to test this hypothesis. The expression of ornithine decarboxylase in mock-transfected COS cells varied depending on the cellular supply of polyamines as has been found in other mammalian cells. Thus, supplementing the cells with exogenous spermidine resulted in a marked decrease in ornithine decarboxylase activity, whereas depletion of intracellular polyamines, using an ornithine decarboxylase inhibitor, gave a large increase in the cellular content of the enzyme. COS cells expressing an ornithine decarboxylase mRNA devoid of its 5' non-translated region did not exhibit any feedback control of the enzyme, neither in the presence of exogenous spermidine nor when the intracellular polyamine levels were depleted to the same extent as in the mock-transfected COS cells. The results strongly suggest that the feedback control of ornithine decarboxylase is not merely a posttranslational phenomenon.

Polyamine-mediated regulation of S-adenosylmethionine decarboxylase expression in mammalian cells. Studies using 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine, a suicide inhibitor of the enzyme.

Cell proliferation is dependent on an adequate supply of the polyamines putrescine, spermidine and spermine. One of the key steps in the polyamine biosynthetic pathway is catalyzed by S-adenosylmethionine decarboxylase (AdoMetDC). In the present study we have used a newly synthesized enzyme-activated irreversible AdoMetDC inhibitor, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine [(Z)-AbeAdo], to investigate the regulation of this enzyme. Treatment of mouse L1210 leukemia cells with (Z)-AbeAdo resulted in a total inhibition of their AdoMetDC activity followed by depletion of the spermidine and spermine content. The putrescine content, however, was dramatically increased after treatment with (Z)-AbeAdo. In spite of the cellular depletion of spermidine and spermine, only a minor inhibitory effect was obtained on cell growth, indicating that putrescine at a high concentration might partly replace spermidine and spermine in their growth-promoting functions. Cells grown in the presence of (Z)-AbeAdo exhibited an increased synthesis of AdoMetDC, which was counteracted by the addition of either spermidine or spermine. The change in AdoMetDC synthesis could not be fully explained by a change in the level of AdoMetDC mRNA, indicating also a translational control. Mammalian AdoMetDC is synthesized as a larger proenzyme, which is then cleaved into two subunits of different sizes. The conversion of the proenzyme into the subunits is a very rapid process, which is stimulated greatly by putrescine in vitro. However, the processing of the proenzyme in the (Z)-AbeAdo-treated L1210 cells was not affected by their very high putrescine content, indicating that the conversion might be saturated at low levels of putrescine, or that most of the putrescine in the (Z)-AbeAdo-treated L1210 cells might be bound to sites normally occupied by spermidine and spermine.

Stabilization of S-adenosylmethionine decarboxylase by aminoguanidine.

Aminoguanidine, an inhibitor of copper-containing amine oxidases, is often used in cell culture studies to prevent the oxidation of added polyamines or their derivatives by the amine oxidase present in bovine serum. However, in the present study, we demonstrate that aminoguanidine, besides affecting the degradation of polyamines, may affect one of the important regulatory enzymes in the biosynthesis of polyamines, namely S-adenosylmethionine decarboxylase (AdoMetDC). Aminoguanidine was shown to induce a 2-3-fold increase in the AdoMetDC levels in L1210 cells. This increase was fully explained by a stabilization of the enzyme, probably caused by the binding of aminoguanidine to the active site of AdoMetDC.

On the translational control of ornithine decarboxylase expression by polyamines.

Ornithine decarboxylase (ODC, EC 4.1.1.17) expression is subject to negative feedback regulation by the polyamines. The results of previous studies favor either translational or post-translational regulation. To facilitate further analysis of the mechanism by which polyamines affect ODC expression we have used a cell line (L1210-DFMOr) that overproduces ODC. This cell line was isolated by selection for resistance to the antiproliferative effect of the ODC inhibitor alpha-difluoromethylornithine (DFMO). These cells respond similarly to polyamine depletion and repletion as do their wild-type counterparts. When L1210-DFMOr cells were grown in the presence of 20 mM DFMO (i.e., when their polyamine content was reduced to an extent that still permitted a normal growth rate) ODC represented 4-5% of the soluble protein synthesized. After transfer of the cells to a medium lacking DFMO (i.e., when their polyamine pools were repleted), the rate of incorporation of [35S]methionine into ODC was one order of magnitude lower. Since this difference in incorporation of radioactivity into ODC remained the same irrespective of the pulse-label time used (between 2 and 20 min) it is likely to represent a true difference in ODC synthesis rate. Consequently, the pulse-label experiments cannot be explained by rapid degradation of the enzyme during the labeling period. The difference in ODC synthesis rate was not accompanied by a corresponding difference in the steady-state level of ODC mRNA. Analyses of the distribution of ODC mRNA in polysome profiles did not demonstrate any major difference between cells grown in the absence or presence of DFMO, even though the ODC synthesis rate differed by as much as 10-fold. However, the distribution of the ODC mRNA in the polysome profiles indicated that the message was poorly translated. Thus, most of the ODC mRNA was present in fractions containing ribosomal subunits or monosomes. Inhibition of elongation by cycloheximide treatment resulted in a shift of the ODC mRNA from the region of the gradient containing ribosomal subunits to that containing mono- and polysomes, indicating that most of the ODC mRNA was accessible to translation. Taken together these data lend support to a translational control mechanism which involves both initiation and elongation.

Regulation of S-adenosylmethionine decarboxylase in L1210 leukemia cells. Studies using an irreversible inhibitor of the enzyme.

A potent irreversible inhibitor of S-adenosylmethionine (AdoMet) decarboxylase, S-(5'-adenosyl)-methylthio-2-aminooxyethane (AdoMeSaoe), was used to study the regulatory control of this key enzyme in the polyamine biosynthetic pathway. Treatment of L1210 cells with the inhibitor completely eradicated the growth-induced rise in AdoMet decarboxylase activity, resulting in a marked decrease in cellular content of spermidine and spermine. The putrescine content, on the other hand, was greatly elevated. Although no detectable AdoMet decarboxylase activity was found in the L1210 cells after treatment with AdoMeSaoe, the cells contained 50-fold higher amounts of AdoMet decarboxylase protein, compared to untreated cells during exponential growth. Part of this increase was shown to be due to elevated synthesis of the enzyme. This stimulation appeared to be related to the decrease in cellular spermidine and spermine content, since addition of either one of the polyamines counteracted the rise in AdoMet decarboxylase synthesis. The synthesis rate was determined by immunoprecipitation of labeled enzyme after a short pulse with [35S]methionine. In addition to a protein that co-migrated with pure rat AdoMet decarboxylase (Mr approximately 32,000), the antibody precipitated a somewhat larger labeled protein (Mr approximately 37,000) that most likely represents the proenzyme form. Treatment of the L1210 cells with AdoMetSaoe also gave rise to a marked stabilization of the decarboxylase which contributed to the increase in its cellular protein content. Addition of spermidine did not significantly affect this stabilization, whereas the addition of spermine reduced the half-life of the enzyme to almost that of the control cells.

Polyamine-mediated control of mammalian S-adenosyl-L-methionine decarboxylase expression: effects on the content and translational efficiency of the mRNA.

The expression of mammalian AdoMet decarboxylase, a key enzyme in polyamine synthesis, was shown to be regulated by polyamines at two different levels. Polyamine depletion of Ehrlich ascites tumor cells induced a marked compensatory increase in the synthesis of the enzyme, as measured by 35S-methionine pulse-labeling and immuno-precipitation. This increase in synthesis rate was counteracted by provision of spermidine, which reduced the synthesis of AdoMet decarboxylase to an undetectable level. Northern analysis revealed a nearly 2-fold increase in the amount of AdoMet decarboxylase mRNA when the putrescine and spermidine content was depleted. This increase in AdoMet decarboxylase mRNA content cannot account for the more than 5-fold increase in synthesis rate, indicating a feedback regulation also at the level of mRNA translation.

Feedback control of ornithine decarboxylase expression by polyamines. Analysis of ornithine decarboxylase mRNA distribution in polysome profiles and of translation of this mRNA in vitro.

Cell growth and differentiation require the presence of optimal concentrations of polyamines. Ornithine decarboxylase (ODC) catalyses the first and rate-controlling step in polyamine synthesis. In studies using cultures of Ehrlich ascites-tumour cells, we have shown that the expression of ODC is subject to feedback regulation by the polyamines. A decrease in the cellular polyamine concentration results in a compensatory increase in the synthesis of ODC, whereas an increase in polyamine concentration results in suppression of ODC synthesis. These changes in ODC synthesis were attributed to changes in the efficiency of ODC mRNA translation, because the steady-state amount of ODC mRNA remained constant. We now show that the number of ribosomes associated with ODC mRNA is low, and that the increase in ODC mRNA translation takes place without a shift in the distribution of ODC mRNA towards larger polysomes. This finding indicates that the polyamines regulate the efficiency of ODC mRNA translation by co-ordinately affecting the rates of initiation and elongation. By analysing ODC mRNA translation in vitro, using a rabbit reticulocyte lysate, polyadenylated RNA from a cell line with an amplified ODC gene, and a monospecific anti-ODC antibody, we also show that spermidine, but not putrescine, exerts a direct regulatory effect on ODC synthesis.