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.     

No role of the 5′ untranslated region of ornithine decarboxylase mRNA in the feedback control of the enzyme

The polyamines are ubiquitous in nature and appear to fulfil several important functions, mostly related to growth, in the cell. The first, and often rate-limiting, step in the biosynthesis of the polyamines is catalysed by ornithine decarboxylase (ODC), which is subject to a variety of control mechanisms. The polyamines exert a strong feedback regulation of the expression - as well as the degradation of the enzyme. The regulation of ODC expression appears to occur at the translational level. The ODC mRNA contains a long GC-rich 5 untranslated region (UTR), which has been demonstrated to hamper the translation of the mRNA. However, it has not yet been conclusively established whether this part of the mRNA fulfils any function in relation to the polyamine-mediated control of ODC synthesis. In the present study, we have used stable transgenic CHO cells, expressing either full-length ODC mRNA or 5 UTR-truncated ODC mRNA, to elucidate the role, if any, of the 5 UTR in the translational regulation of the enzyme by polyamines. No differences in regulatory properties were observed between the cells expressing the full-length ODC mRNA and those expressing the ODC mRNA devoid of most the 5 UTR. The cell lines down-regulated ODC (synthesis as well as activity) to the same extent upon exposure to an excess of polyamines, demonstrating that the feedback control of ODC mRNA translation occurs by a mechanism independent of the major part of the 5 UTR of the ODC mRNA.     

Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.     

Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5' leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiations. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.     

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.     

Feedback regulation of ornithine decarboxylase expression. Studies using a polysomal run-off system

The rate-controlling enzyme in polyamine synthesis, ornithine decarboxylase (ODC), is subject to feedback regulation by the polyamines at the level of translation. In the present study we used a cell-free translation system to further investigate the mechanism by which this regulation occurs. Lysates of ODC-overproducing cells were capable of synthesizing large amounts of ODC. The degree of initiation was poor in the lysates and the synthesis of ODC was mainly a result of continued elongation of peptide chains on pre-initiated ribosomes. By determining the amount of ODC produced in the lysate, we obtained an estimate of the number of ribosomes that were actively translating ODC mRNA at the moment of lysis. Using this polysomal run-off assay we demonstrated that the polyamine-mediated regulation of ODC synthesis occurs without any change in the number of ribosomes associated with the message. This finding indicates that the polyamines exert a coordinate effect on initiation and elongation.     

Control of ornithine decarboxylase activity in alpha-difluoromethylornithine-resistant L1210 cells by polyamines and synthetic analogues

The regulation of ornithine decarboxylase (ODC) activity by the polyamine derivatives N1,N8-bis(ethyl)-spermidine and N1,N12-bis(ethyl)spermine was studied using a line of L1210 cells resistant to alpha-difluoromethylornithine (D-R cells), which contain very high levels of ODC, and a synthetic mRNA prepared from a plasmid containing an insert corresponding to ODC mRNA adjacent to an SP6 RNA polymerase promoter. Studies in which ODC protein was labeled in the D-R cells by exposure to [35S]methionine indicated that the polyamine derivatives and their physiological counterparts led to an increased rate of degradation of ODC and to a rapid reduction in ODC synthesis without affecting the content of ODC mRNA. Direct evidence that the polyamine derivatives act by inhibiting the translation of the ODC mRNA was obtained by studying their effects on the translation of ODC mRNA in reticulocyte lysates. This translation was strongly inhibited by the addition of N1,N8-bis(ethyl)spermidine, spermidine, N1,N12-bis(ethyl)spermine, or spermine but was not affected much by putrescine. The inhibition of the translation of ODC mRNA by either of the bis(ethyl) polyamine derivatives occurred at concentrations which stimulated total protein synthesis showing the selectivity of the reduction in ODC. The effects of polyamine derivatives and polyamines on translation of the plasmid-derived ODC mRNA were identical with those found with the D-R L1210 cell mRNA. This synthetic ODC mRNA lacks 261 bases of the 5'-leader sequences and 200 bases plus the poly(A) section from the 3'-nontranslated sequence. Therefore, these regions appear not to influence sensitivity of the ODC mRNA to inhibition of translation by polyamine derivatives.     

Regulated degradation of ornithine decarboxylase requires interaction with the polyamine-inducible protein antizyme.

Intracellular degradation of vertebrate ornithine decarboxylase (ODC) is accelerated by polyamines, the products of the pathway controlled by ODC. Antizyme, a reversible, tightly binding protein inhibitor of ODC activity, is believed to be involved in this process. Mouse and Trypanosoma brucei ODCs are structurally similar, but the trypanosome enzyme, unlike that of the mouse, is not regulated by intracellular polyamines when expressed in hamster cells (L. Ghoda, D. Sidney, M. Macrae, and P. Coffino, Mol. Cell. Biol. 12:2178-2185, 1992). We found that mouse ODC interacts with antizyme in vitro but trypanosome ODC does not. To localize the region necessary for binding, we made a series of enzymatically active chimeric mouse-trypanosome ODCs and tested them for antizyme interaction. Replacing residues 117 to 140 within the 461-amino-acid mouse ODC sequence with the equivalent region of trypanosome ODC disrupted both antizyme binding and in vivo regulation. Formation of an antizyme-ODC complex is therefore required for regulated degradation.     

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.     

Intracellular localization of ornithine decarboxylase and its regulatory protein, antizyme-1.

The enzyme ornithine decarboxylase (ODC) and its regulatory protein antizyme-1 (AZ1) are key regulators in the homeostasis of polyamines. To gain more insight into the exact intracellular distribution of ODC and AZ1, we performed immunocytochemical and Green Fluorescent Protein-fluorocytochemical studies in cultured human cervix carcinoma and human prostatic carcinoma (PC-346C) cells. ODC localization patterns varied from predominantly cytoplasmic to both cytoplasmic and nuclear staining, whereas AZ1 was mostly found in the nucleus. In cells that were synchronized in the mitotic phase, localization of both ODC and AZ1 changed from perinuclear at the beginning of mitosis into nucleoplasmic at close proximity to the chromosomes during meta-, ana- and telophase. Upon completion of mitosis, localization of ODC and AZ1 was reverted back to the cytoplasm, i.e., predominantly perinuclear immediately after cytokinesis. When PC-346C cells were treated with polyamines to induce AZ1-regulated ODC degradation, ODC was predominantly found in the nucleus and colocalized with immunoreactive AZ1. A comparable accumulation of ODC and AZ1 in the nucleus was found in PC-346C cells treated with the polyamine analog SL-11093. The present study suggests that AZ1 is involved in nucleocytoplasmic shuttling of ODC, which may be a prerequisite for ODC regulation and/or function.     

A mammalian sequence-dependent upstream open reading frame mediates polyamine-regulated translation in yeast

In mammals, control of S-adenosylmethionine decarboxylase (AdoMetDC) translation is one component of a feedback network that regulates intracellular levels of the polyamines, spermidine, and spermine. AdoMetDC mRNA from mammals contains a highly conserved upstream open reading frame (uORF) within its leader sequence that confers polyamine-regulated suppression of translation on the associated downstream cistron. This regulation is mediated through an interaction that depends on the amino acid sequence of the uORF-encoded hexapeptide. It remains to be shown whether polyamines participate directly in this interaction or indirectly through a specialized signal transduction pathway. We show that Saccharomyces cerevisiae does not have a uORF associated with its AdoMetDC gene (SPE2) and that ribosome loading on the SPE2 mRNA is not positively influenced by polyamine depletion, as it is in mammalian cells. Nevertheless, the mammalian AdoMetDC uORF, when introduced into a polyamine auxotroph of yeast, conferred polyamine regulation of both translational efficiency and ribosome loading on the associated mRNA. This regulatory activity depended on the amino acid sequence encoded by the fourth and fifth codons of the uORF, as in mammalian cells. The fact that the regulatory properties of this mammalian translational control element are quite similar in both mammalian and yeast cells suggests that a specialized signal transduction pathway is not required. Rather, it seems likely that polyamines may be directly participating in an interaction between the uORF-encoded peptide and a constitutive component of the translation machinery, which leads to inhibition of ribosome activity.     

Destabilization of ornithine decarboxylase by transfected antizyme gene expression in hepatoma tissue culture cells.

The degradation of ornithine decarboxylase (ODC) is stimulated by polyamines in a protein synthesis-dependent manner. It has been suggested that antizyme, an ODC-inhibiting protein induced by polyamines, is involved in the process of polyamine-stimulated ODC decay. In this study, we investigated the direct effect of antizyme on ODC decay in hepatoma tissue culture (HTC) cells. A truncated rat antizyme cDNA, Z1, was inserted into an expression vector at a site under the control of a glucocorticoid-inducible promoter and transfected into HTC cells. In the transfected cells dexamethasone increased the amount of Z1 mRNA and induced active antizyme in the absence of exogenous polyamines. When dexamethasone was added to cells with a high level of ODC, rapid decays of ODC activity and protein were elicited after a lag time. Cycloheximide abolished the effect of dexamethasone. These effects of dexamethasone were not observed in control HTC cells transfected with the chloramphenicol acetyltransferase gene. This study indicated that, once induced, antizyme stimulated ODC degradation independently of polyamines and strongly supported our previous hypothesis that the ODC decay-accelerating action of polyamines is mediated by antizyme.     

Expression and post-transcriptional regulation of ornithine decarboxylase during early Xenopus development.

In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred during early Xenopus development. Following fertilization, this enzyme activity rises with a quantitatively correlated accumulation of putrescine and spermidine. This increase in ODC activity was associated with an increased translation of the maternal ODC mRNA, which was stable in the embryo and whose polyadenylation increased slightly between fertilization and the mid-blastula transition (MBT). ODC activity was stable in cycloheximide-treated embryos, indicating that before the MBT this enzyme was not degraded. After the MBT, ODC activity fell, but no decrease in this mRNA was observed. In gastrulae, ODC mRNA was both increased in amount and polyadenylated. The reduced ODC activity at this stage of development was not associated with a fall in ribosome loading of the mRNA. Treatment of post-MBT embryos with cycloheximide lead to an accentuation of the normally observed decrease in ODC activity. Expression of Xenopus ODC in mutant ODC-deficient Chinese hamster ovary cells (C 55.7 cells) showed that the Xenopus enzyme was rapidly degraded and can be regulated post-translationally by polyamines, indicating that the post-MBT fall in ODC activity could be caused by a change in protein turnover or by polyamine-mediated regulation.     

The development of competence in resting B cells. The induction of cyclic AMP and ornithine decarboxylase activity after direct contact between B and T helper cells.

In the present communication, an experimental approach is utilized that facilitates the study of biochemical processes induced in B cells after their interaction with Th cells. In this approach, Th cell clones are stimulated for 18 h upon anti-CD3-coated plates, fixed with paraformaldehyde, and added at a 2 to 3:1 ratio to small, resting B cells (isolated from Percoll gradients). Th cells not stimulated on anti-CD3-coated plates, but fixed with paraformaldehyde, serve as controls for these experiments. The activated, fixed Th cells induce a transient, sixfold increase in B cell levels of cAMP, as well as an increase in B cell expression of ornithine decarboxylase (ODC) activity. This enzyme initiates the synthesis of polyamines and has been shown to be increased as cells enter the growth phase. In addition, previous studies have shown that the cellular levels of ODC activity are controlled by a multi-tiered regulatory cascade. To examine this aspect, polyclonally stimulated B cells were studied. Such cells demonstrated a gradual increase in ODC mRNA levels that peaked between 6 and 15 h and can be partially explained by a three- to fourfold increase in mRNA stability but not by changes in the enzyme affinity for substrate. The increase in ODC mRNA occurs in the absence of protein synthesis, suggesting that the ODC gene is a member of the immediate/early gene family. Finally, the early increase in ODC mRNA was enhanced in cells in which cAMP levels were artificially elevated, suggesting the possibility that the cAMP-dependent signaling pathway participates during the regulation of this gene expression. The significance of these experimental results concerning the process of B cell activation is discussed.