Olle Heby

Translational regulation of ornithine decarboxylase by polyamines

The activity of ornithine decarboxylase (ODC), the first and rate‐limiting enzyme in the polyamine biosynthetic pathway, is dramatically increased in proliferating cells. In addition to transcriptional regulation of ODC, the present study shows that the enzyme is regulated at the translational level by putrescine and spermidine. ODC synthesis is inhibited by an increase and stimulated by a decrease in their cellular content. Spermidine is a more potent negative regulator than is putrescine. The effects of polyamines on ODC synthesis were not attributable to changes in the cellular content of ODC mRNA, thus demonstrating regulation at the translational level.

Ornithine decarboxylase inhibitors increase the cellular content of the enzyme: implications for translational regulation.

Ehrlich ascites tumor cells grown in the presence of inhibitors of ornithine decarboxylase (EC 4.1.1.17) exhibited an elevated content of this enzyme. The increase could not solely be explained by a decrease in the degradation rate of the enzyme. Instead a stimulation of enzyme synthesis, probably mediated via the polyamine-depleting properties of the inhibitors, is suggested. The enhancement of cellular ornithine decarboxylase content was not accompanied by any significant changes in the amount of ornithine decarboxylase mRNA, indicating a regulation at the level of translation.

Polyamines in early embryonic development: Their relationship to nuclear multiplication rate, cell cycle traverse, and nucleolar formation in a dipteran egg☆

Polyamine synthesis and accumulation were assessed from fertilization until gastrulation in a dipteran egg (Calliphora erythrocephala Meigen). Spermidine synthesis was activated immediately after fertilization, generating a broad spermidine peak during early cleavage. This period is characterized by the most rapid nuclear multiplication known from animal material. Cleavage consists of nuclear multiplication only, and the egg remains syncytial until gastrulation. After nine synchronous nuclear divisions with a cycle length of 10 min, the cycle length is gradually increased to 20 min during the subsequent four parasynchronous nuclear divisions. The spermidine level decreased in parallel with this decreasing rate of nuclear division. The interphase of the next nuclear cycle is remarkably prolonged and lasts for more than 90 min, i.e., until after the onset of gastrulation. It consists of an initial short S phase followed by a longer G2 phase; G1 is extremely short or absent. During this prolonged interphase, spermidine content showed a biphasic pattern of changes with peaks during S and late G2. The S-phase peak also coincides with the first appearance of nucleoli during embryogenesis. The late-G2-phase peak coincides with the period of rapid cytokinesis, during which all nuclei in the peripheral layer of the syncytium become separated by membranes forming a cellular blastoderm. The polyamine pattern is consistent with the idea that the polyamines play an important role in DNA replication and in cytokinesis as well as in nucleolar formation.

Irreversible inhibition of the early increase in ornithine decarboxylase activity following growth stimulation is required to block Ehrlich ascites tumor cell proliferation in culture

Summary Ehrlich ascites tumor cell growth in culture was inhibited by α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, provided that treatment was initiated at the time of growth stimulation. When α-difluoromethylornithine was added after the activation of polyamine synthesis caused by the growth stimulus, i.e. when a 3–5-fold increase in putrescine and spermidine content had occurred, cell proliferation was completely unaffected. α-Methylornithine, a competitive inhibitor of ornithine decarboxylase, did not affect cell proliferation even when added at the time of growth stimulation. Compared to α-difluoromethylornithine, α-methylornithine only produced a partial and transient decrease in the cellular putrescine and spermidine content.

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.

Ornithine decarboxylase as target of chemotheraphy

Ornithine decarboxylase is a key enzyme in polyamine synthesis and growth of mammalian cells. In this chapter I review recent reports on the purification and properties of the pure enzyme, and on the localization, synthesis and regulation of the enzyme in the cell. The use of monospecific antibodies, radiolabeled irreversible inhibitors and cDNA clones for studying enzyme localization, turnover and regulation, is briefly described. This first part is meant to serve as a basis for the analysis of ornithine decarboxylase as a target of chemotherapy. A selection of the most potent inhibitors of ornithine decarboxylase is presented and the effects of some of these in cell culture, in animals and in the clinical setting are reviewed.

Simplified micro-method for the quantitative analysis of putrescine, spermidine and spermine in urine

A simplified micro-method for the quantitative analysis of urinary polyamines is described. After acid hydrolysis of urine, the polyamines are converted to fluorescent 1-dimethylaminonaphthalene-5-sulfonyl (Dns; dansyl) derivatives and separated by means of thin-layer chromatography. Dns-NH2, which has been reported to interfere with the determination of putrescine, is well separated from di-Dns-putrescine. Putrescine, spermidine and spermine are quantitated by in situ scanning of their fluorescent spots on the chromatogram. The present method is both sensitive and reproducible. It eliminates a number of time-consuming steps and thus reduces preparative losses. Yet an adequate chromatographic resolution is obtained. Representative polyamine analyses of urine from normal volunteers and from cancer patients are reported. Elevated levels occur in the urines of pregnant women and of patients with various types of cancer.

Polyamines, DNA Methylation and Cell Differentiation

The cellular concentration of AdoMet is normally very much higher than that of dcAdoMet, the aminopropyl group donor in polyamine synthesis. However, when cells are depleted of their putrescine and spermidine, i.e. the aminopropyl group acceptors, the dcAdoMet concentration increases dramatically, to a level that may greatly exceed that of AdoMet. Using a highly purified DNA methyltransferase and its preferred substrates, a defined hemimethylated duplex oligodeoxynucleotide or poly(dI-dC)-poly(dI-dC), we demonstrate that dcAdoMet is a poor methyl group donor, and that it starts to inhibit DNA methylation when its concentration exceeds that of AdoMet. At a dcAdoMet/AdoMet ratio of 5:1 there is very little methyl transfer. This study suggests that the antiproliferative and differentiative effects brought about by inhibitors of polyamine synthesis may be partly attributable to dcAdoMet-mediated inhibition of DNA methylation.

Effects of polyamine limitation on nucleolar development and morphology in early chick embryos.

Inhibition of polyamine synthesis in early chick embryos by in ovo treatment with DL-alpha-difluoromethylornithine (DFMO), injected beneath the blastoderm after 5 h of incubation, permanently blocks post-gastrular development. After the first day of polyamine limitation, the embryos possess a thickened primitive streak. Further morphogenesis is blocked and the ectoderm and mesoderm are condensed around the streak. There is obvious suppression of nucleolar formation in 24 h DFMO-treated embryos. In the mesoderm obliquely in front of Hensens node the frequency of nucleolus-possessing cells is only a few percent lower in DFMO-treated than in control embryos. However, in the same area the frequency of mesoderm cells possessing multiple nucleoli is about 50% lower in the polyamine-depleted embryos. At the ultrastructural level, mesoderm cells from 24 h DFMO-treated embryos show a reduction of the fibrillar component of the nucleolus with a resulting segregation of the nucleolar material. Our data indicate that stimulation of polyamine synthesis is an obligatory step in the differentiation of epiblast cells into mesoderm cells.

Polyamine biosynthetic enzymes as targets in cancer chemotherapy

In this chapter we focus attention on recent developments in the biosynthesis of putrescine, spermidine and spermine and their linkage to salvage pathways of methionine and adenine nucleotide synthesis. We describe the use of specific inhibitors of the polyamine biosynthetic enzymes for studying the role of polyamines in cell growth and division as well as in cell differentiation. Some novel findings are presented which suggest that part of the inhibitory action that polyamine synthesis inhibitors exert on DNA synthesis may be due to the accumulation of ADP and ATP. We show that polyamine synthesis inhibitors are capable of inducing terminal differentiation of neoplastic cells to forms with no further proliferative potential, and briefly discuss the potential use of this approach in cancer chemotherapy.