Ingvar Holm

Interference with DNA Methyltransferase Activity and Genome Methylation during F9 Teratocarcinoma Stem Cell Differentiation Induced by Polyamine Depletion

When ornithine decarboxylase, the initial and highly regulated enzyme in polyamine biosynthesis, is irreversibly inactivated by α-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.

Expression of transforming growth factor-βI in rat ventral prostate and Dunning R3327 PAP prostate tumor after castration and estrogen treatment

In normal prostate, TGF‐β1 is associated to castration induced apoptosis. Combined castration and estrogen treatment, but not castration alone, induces apoptosis in the Dunning R3327 PAP adenocarcinoma.

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.

EFFECTS OF CHRONICALLY ELEVATED GROWTH HORMONE LEVELS ON POLYAMINE METABOLISM IN ELDERLY TRANSGENIC MICE

The polyamines are ubiquitous, multifunctional aliphatic amines with roles in cell growth, proliferation, differentiation, and malignant development. After growth stimulation, rapid and transient changes occur in polyamine regulatory enzymes. In this respect, acute effects of growth hormone (GH) injection on polyamine metabolic enzymes have earlier been shown. The present investigation comprises studies of the effects on polyamine metabolism of constitutively elevated levels of circulating GH in elderly transgenic (tg+) mice, overexpressing bovine GH. Polyamine levels were found to be constitutively altered in the liver and kidney of tg+ mice. Less changes were found in the spleen and none in the brain. The cellular uptake of polyamines in the liver from tg+ mice showed an increase and considerable changes were observed in the activity of ornithine decarboxylase (ODC) in the liver and kidney and S-adenosylmethionine decarboxylase (AdoMetDC) in the liver. A conspicuous finding was the distribution pattern of ODC protein in the liver and both tg- and tg+ animals. The results show that the effects of chronically elevated GH levels are organ-dependent and complex, and differ from acute GH effects. Despite high ODC activity and polyamine levels in liver, these mice did not display any malignant transformation even at an advanced age, indicating that high ODC activity is not sufficient to induce tumorigenesis in vivo.

DOWN-REGULATION OF ORNITHINE DECARBOXYLASE BY AN INCREASED DEGRADATION OF THE ENZYME DURING GASTRULATION OF XENOPUS LAEVIS

The present study was designed to analyze the regulation of the levels of the polyamines and their biosynthetic enzymes during embryonic development of Xenopus laevis. The activity of ornithine decarboxylase (ODC), a rate-controlling enzyme in polyamine biosynthesis, is elevated until, during gastrulation, there is a precipitous drop in activity. This is not attributable to a decrease in ODC mRNA content and polysome profiles reveal no apparent decrease in ODC message associated with polysomes. ODC synthesis seems to be maintained at a low, relatively constant rate until neurulation whereupon ribosome loading of ODC mRNA increases. During gastrulation the rate of ODC degradation increases dramatically, which can account for the decrease in ODC. S-Adenosylmethionine decarboxylase (AdoMetDC), another rate-controlling enzyme in polyamine biosynthesis, shows a low and constant activity from cleavage to neurulation. Subsequently, the AdoMetDC activity increases dramatically. The changes in AdoMetDC activity parallel the changes in AdoMetDC mRNA levels, suggesting a transcriptional control of AdoMetDC expression during this development period. The activities of ODC and AdoMetDC produce a steady increase in putrescine and spermidine content of the embryo. The spermine content also increases until gastrulation, but then decreases until the tailbud stage.

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.