Maria Halmekytö

Enhanced papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene

We have studied the induction of papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene and in their nontransgenic littermates. The transgenic animals displayed a basal epidermal ornithine decarboxylase activity that was nearly 20 times higher than in their nontransgenic littermates. A single topical application of 12-O-tetradecanoylphorbol-13-acetate induced a much more profound and longer-lasting increase in transgene-derived ornithine decarboxylase activity in comparison with the endogenous enzyme activity. Initiation of skin tumorigenesis with a single topical application of dimethylbenz[a]antracene followed by twice-weekly application of 12-O-tetradecanoylphorbol-13-acetate resulted in the appearance of first papillomas both in nontransgenic and transgenic animals by week 7. However, after 11 weeks of 12-O-tetradecanoylphorbol-13-acetate application, the number of papillomas per animal was almost 100% higher in the transgenic animals than in their nontransgenic littermates. These results indicate that an overexpression of epidermal ornithine decarboxylase confers a growth advantage on skin tumors in vivo.

Cerebral Energy Metabolism and Immediate Early Gene Induction Following Severe Incomplete Ischaemia in Transgenic Mice Overexpressing the Human Ornithine Decarboxylase Gene: Evidence that Putrescine is not Neurotoxic In Vivo

Cerebral ischaemia causes activation of ornithine decarboxylase followed by accumulation of putrescine, and these biochemical phenomena have been thought to contribute to the development of neuronal damage. We have used a transgenic mouse line overexpressing the human ornithine decarboxylase gene in their neurons with constitutively high putrescine to study the possible role of putrescine in development of neuronal damage in forebrain ischaemia. An incomplete forebrain ischaemia model was developed in which common carotid arteries were bilaterally occluded and reduction of blood pressure caused by orthostatic reaction was used as a way of decreasing cerebral circulation. Cerebral high‐energy metabolites, intracellular pH and lactate were monitored by means of 31P and 1H nuclear magnetic resonance spectroscopy respectively. Incomplete ischaemia for 15 min resulted in severe energy failure, as indicated by an increase in the inorganic phosphate/phosphocreatine ratio, intracellular acidification from a pH of ∼7.1 to ∼6.5 and an increase in lactate concentration from <1 to ∼10 mmol/kg in both syngenic and transgenic mice. Following deocclusion, recovery of energy metabolites, intracellular pH and lactate were identical in both animal groups. Ornithine decarboxylase activity rose 9‐ and 3‐fold in syngenic and transgenic mice respectively 6 h after ischaemia. Activation of ornithine decarboxylase resulted in extensive accumulation of putrescine in the brains of transgenic animals 12–24 h after ischaemia, which was ∼50‐fold greater than the basal level in syngenic mice. In situ hybridization experiments revealed induction of transcription factors c‐Fos and zif‐268 in the hippocampus, throughout the cerebral cortex and striatum 1–3 h after ischaemia. Messenger RNA of heat shock protein 70 was induced in dentate gyrus and CA3 and CA4 subfields of the hippocampus 1 h after ischaemia. The distribution and extent of induced mRNAs were similar in syngenic and transgenic animals. Histological evaluation did not reveal any difference between the two animal groups despite large variation in their cerebral putrescine content. Neuronal necroses were observed in the CA4 layer of hippocampi in both syngenic and transgenic mice. These data suggest that ornithine decarboxylase activation and accumulation of endogenous putrescine are indicative of an ischaemic insult and that these changes reflect an adaptive response rather than acting as causative factors of neuronal damage.

Nuclear Magnetic Resonance Spectroscopy Study on Energy Metabolism, Intracellular pH, and Free Mg2+ Concentration in the Brain of Transgenic Mice Overexpressing Human Ornithine Decarboxylase Gene

Abstract: We have generated a transgenic mouse line strikingly overexpressing the human ornithine decarboxylase (ODC) gene in their brain. Brain ODC activity was increased in the transgenic animals by a factor of 70 in comparison with their nontransgenic littermates. The content of brain putrescine, the product of ODC, was >60 μmol/g of tissue in the transgenic mice, whereas in the normal animals it was below the level that could be detected by an HPLC method. The concentrations of the higher polyamines (spermidine and spermine) were not significantly different from control values. 31P nuclear magnetic resonance (31P NMR) Spectroscopy analyses revealed a significantly reduced (40%) free Mg2+ concentration as calculated from the chemical shift differences of the nucleoside triphosphate a and ft peaks in the brains of the transgenic animals. The lower free Mg2+ concentration in the brains of ODC transgenic mice was not a consequence of altered intracellular pH or changes in cellular high‐energy metabolites. 1H NMR showed no differences in brain choline/N‐acetylaspartate and total creatine/N‐acetylaspartate ratios between the two animal groups. These ODC transgenic animals may serve as models in vivo for studies on cerebral postischemic events and on epilepsy, as polyamines are supposed to be involved in these processes.

Methylation of human ornithine decarboxylase gene before transfection abolishes its transient expression in Chinese hamster ovary cells

Different methylations of cloned human ornithine decarboxylase gene with restriction methylases in vitro before transfection greatly reduced the transient expression of ODC in Chinese hamster ovary cells. Single methylation of the gene with Hpa II (CCGG) methylase decreased the transiently expressed peak activity by about 50%, single methylation with Hha I (CCGG) methylase by about 80% whilst a double methylation at both Hpa II and Hha I restriction sites virtually abolished any transiently expressed ornithine decarboxylase activity. These results together with our earlier circumventing evidence indicate that the expression of mammalian ornithine decarboxylase is critically influenced by the methylation state of the gene.