No abstract

We have produced several transgenic mouse lines over-expressing the human ornithine decarboxylase (ODC) gene. We have now characterized one of the transgenic lines as regards the tissue accumulation of the polyamines and the activities of their metabolizing enzymes. Among the tissues analysed, the polyamine pattern was most strikingly changed in testis and brain of the transgenic animals. ODC activity was greatly enhanced in all tissues, except kidney, of the transgenic animals. The most dramatic increase, 80-fold, was found in brain of the transgenic mice. The activities of S-adenosylmethionine decarboxylase and spermidine and spermine syntheses were likewise significantly increased in testis of the transgenic animals. The activities of the enzymes involved in the back-conversion of the polyamines, namely spermidine/spermine acetyltransferase and polyamine oxidase, were similar in the transgenic and non-transgenic animals. As analysed by reverse transcriptase/polymerase chain reaction, all the six tissues of the transgenic animals expressed human-specific ODC mRNA. Determination of the half-life of testicular ODC revealed a stabilization of the enzyme in the transgenic males.

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The hepatic synthesis and accumulation of S-adenosylhomocysteine, S-adenosylmethionine and polyamines were studied in normal and vitamin B-6-deficient male albino rats. A method involving a single chromatography on a phosphocellulose column was developed for the determination of S-adenosylhomocysteine and S-adenosylmethionine from tissue samples. Feeding the rat with pyridoxine-deficient diet for 3 or 6 weeks resulted in a four- to five-fold increase in the concentration of S-adenosylhomocysteine, whereas that of S-adenosylmethionine was only slighly elevated. The concentration of putrescine was decreased to half, that of spermidine was somewhat decreased and that of spermine remained fairly constant. The activities of L-ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, L-methionine adenosyltransferase and S-adenosyl-L-homocysteine hydrolase were moderately increased. S-Adenosylmethionine decarboxylase showed no requirement for pyridoxal 5'-phosphate. The major effect of pyridoxine deficiency of S-adenosylmethionine metabolism seems to be a block in the utilization of S-adenosylhomocysteine, resulting in the accumulation of this metabolite to a concentration that may inhibit biological methylation reactions.

Polyamines are known to be essential for normal cell growth and differentiation. However, despite numerous studies, specific cellular functions of polyamines in general and individual polyamines in particular have remained only tentative, because of a lack of appropriate cell lines in which genes of polyamine-synthesizing enzymes have been disrupted by gene targeting. With the use of homologous recombination technique, we disrupted the gene encoding spermine synthase in mouse embryonic stem cells. The spermine synthase gene is located on X chromosome in mouse and, because the cells used in this study were of XY karyotype, a single targeting event was sufficient to result in null genotype. The targeted cells did not have any measurable spermine synthase activity and were totally devoid of the polyamine spermine. Spermine deficiency led to a substantial increase in spermidine content, but the total polyamine content was nearly unchanged. Despite the lack of spermine, these cells displayed a growth rate that was nearly similar to that of the parental cells and showed no overt morphological changes. However, the spermine-deficient cells were significantly more sensitive to the growth inhibition exerted by 2-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Similarly, methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, and diethylnorspermine, a polyamine analog, although exerting cytostatic growth inhibition on wild-type cells, were clearly cytotoxic to the spermine-deficient cells. The spermine-deficient cells were also much more sensitive to etoposide-induced DNA damage than their wild-type counterparts.