When ornithine decarboxylase, the initial and highly regulated enzyme in polyamine biosynthesis, is irreversibly inactivated by ␣-difluoromethylornithine, F9 teratocarcinoma stem cells are depleted of putrescine and spermidine and as a result differentiate into a cell type which phenotypically resembles the parietal endoderm cells of the early mouse embryo. Simultaneously the level of decarboxylated S-adenosylmethionine (dcAdoMet), the aminopropyl group donor in spermidine and spermine synthesis, increases dramatically, as the aminopropyl group acceptor molecules (putrescine and spermidine) become limiting. When this excessive accumulation of dcAdoMet is prevented by specific inhibition of the AdoMet decarboxylase activity, the differentiative effect is counteracted, despite the fact that the extent of polyamine depletion remains almost identical. Therefore, it may be concluded that dcAdoMet plays an important role in the induction of differentiation. Moreover, this key metabolite acts as a competitive inhibitor of DNA methyltransferase and is therefore capable of interfering with the maintenance methylation of newly replicated DNA. During the course of F9 cell differentiation, the highly methylated genome is gradually demethylated, and its pattern of gene expression is changed. Our present findings, that the DNA remains highly methylated and that the differentiative process is counteracted when the build-up of dcAdoMet is prevented, provide strong evidence for a causative relation between the level of dcAdoMet and the state of DNA methylation as well as cell differentiation.Cells require optimal levels of the polyamines, putrescine, spermidine, and spermine, for their growth and differentiation and are therefore equipped with many intricate mechanisms for the control of these levels (1, 2). The polyamine biosynthetic pathway consists of two highly regulated enzymes, ornithine decarboxylase (ODC) 1 and S-adenosylmethionine decarboxylase (AdoMetDC), and two constitutively expressed enzymes, spermidine synthase and spermine synthase (Fig. 1). Any cell that becomes polyamine-deficient is severely limited in its ability to grow and proliferate. There is also evidence suggesting that polyamines play a role in apoptotic cell death (3, 4). More specifically, the polyamines have been shown to regulate ion channel gating (5-7) and polyamine derivatives are constituents of venoms produced by many invertebrates (e.g. funnel-web spiders), where they act as potent neurotoxins (8). Polyamines also regulate programmed translational frameshifting, which is essential for the expression of ODC antizyme, a protein involved in the regulation of polyamine levels both in yeast (9) and in mammalian (10) cells.Of particular interest, with regard to growth regulation, is the finding that when ODC genes are transfected into cells and overexpressed, the cells go through malignant transformation (11). The fact that inhibition of the ODC activity counteracts malignant transformation suggests that the ODC gene is a proto-oncogene. In this co...

The ornithine decarboxylase (ODC) gene is a transcriptional target of c-Myc. Exponentially growing cells usually exhibit high c-Myc levels and high ODC levels, whereas stationary phase cells and terminally differentiated cells have low levels of both proteins. Therefore, we were surprised to find that when F9 teratocarcinoma stem cells were blocked in the G(1) phase of their cell cycle and induced to differentiate by irreversible inhibition of the ODC activity, the expression of c-Myc was up-regulated instead of being down-regulated. During the course of differentiation, the c-myc gene was constitutively expressed, and c-Myc protein accumulated. In transfection experiments, using ODC promoter-reporter gene fusion constructs, the accumulation of c-Myc protein, resulting from polyamine depletion, led to increased reporter gene expression. This finding is consistent with the view that depletion of polyamines relieves the suppression that they exert on c-myc mRNA translation, causing an accumulation of c-Myc protein, which in turn transactivates its target gene, the bona fide ODC gene. Thus, the accumulation of an active c-Myc protein does not preclude differentiative events, nor does it override the growth arrest caused by polyamine depletion. These results suggest a new role for polyamines-as negative regulators of c-Myc expression.