We have generated a transgenic mouse line that overexpresses the rate-controlling enzyme of polyamine catabolism, spermidine/spermine N 1 -acetyltransferase. Tissues of these mice showed markedly distorted polyamine pools, which in most cases were characterized by the appearance of N 1 -acetylspermidine, not normally found in mouse tissues, a massive accumulation of putrescine, and decreases in spermidine and/or spermine pools. The most striking phenotypic change was permanent hair loss at the age of 3 to 4 weeks which was typified histologically by the appearance of extensive follicular cysts in the dermis. The effect seemed attributable to putrescine interference with hair development, possibly with differentiation/proliferation of epidermal cells located in hair follicles. Female members of the transgenic line were found to be infertile apparently due to ovarian hypofunction and hypoplastic uteri. The findings demonstrate the utility of spermidine/spermine N 1 -acetyltransferase overexpression as an effective means for genetically modulating total tissue polyamine pools in transgenic animals and examining the developmental and oncogenic consequences.The well recognized association of polyamines with cell growth (1-3) is best illustrated by findings related to the key polyamine biosynthetic enzyme, ornithine decarboxylase (ODC).1 Although ODC is sharply but transiently increased by growth stimuli, it is constitutively activated during cell transformation induced by carcinogens, viruses, or oncogenes. Overexpression of ODC has been correlated with increased proliferative potential (4), tissue invasiveness (5), and in certain cell types, with oncogene-like transforming capabilities (6 -8). Thus far, ODC appears to be the only growth-related gene activated by the transcription factors c-myc (9 -11) and n-myc (12), suggesting a critical role for the enzyme in growth control. However, as indicated below, findings obtained in cell culture may not be directly applicable to conditions prevailing in vivo.To define the role of polyamines in proliferative processes associated with the whole animal, we have generated a number of transgenic mouse and rat lines that overexpress ODC and/or other polyamine biosynthetic enzymes. Given the importance of polyamine biosynthetic activity to cell growth, the phenotypic changes were unexpectedly mild. In transgenic mice overexpressing ODC, the most marked effect was inhibition of meiotic DNA synthesis during spermatogenesis (13) ultimately leading to male infertility (14). It is also noteworthy that lifelong overexpression of ODC in mouse tissues did not seem to increase the incidence of spontaneous tumors (15). The absence of more profound phenotypic changes in these mice may be attributable to the relatively minor changes observed in higher polyamine pools. Despite severalfold increases in ODC activity, polyamine pool disturbances were mainly confined to putrescine accumulation, and the pools of those polyamines considered to be more significantly involved in cell growth, spermidi...
Acetylation of polyamines by spermidine/spermine N 1 -acetyltransferase (SSAT) has been implicated in their degradation and/or export out of the cell. The relationship of SSAT to polyamine pool dynamics and cell growth is not yet clearly understood. MCF-7 human breast carcinoma cells were transfected with tetracycline-regulated (Tet-off) SSAT human cDNA or murine gene. Doxycycline removal for >2 days caused a ϳ20-fold increase in SSAT RNA and a ϳ10-fold increase in enzyme activity. After 4 days, intracellular putrescine and spermidine pools were markedly lowered, and cell growth was inhibited. Growth inhibition could not be prevented with exogenous polyamines due to a previously unrecognized ability of SSAT to rapidly acetylate influxing polyamines and thereby prevent restoration of the endogenous pools. Instead, cells accumulated high levels of N 1 -acetylspermidine, N 1 -acetylspermine, and N 1 ,N 12 -diacetylspermine, a metabolite not previously reported in mammalian cells. Doxycycline deprivation before treatment with N 1 ,N 11-diethylnorspermine markedly increased analog induction of SSAT mRNA and activity and enhanced growth sensitivity to the analog by ϳ100-fold.Overall, the findings demonstrate that conditional overexpression of SSAT lowers polyamine pools, inhibits cell growth, and markedly enhances growth sensitivity to certain analogs. The enzyme also plays a remarkably efficient role in maintaining polyamine pool homeostasis during challenges with exogenous polyamines.Intracellular polyamine pools appear to be sensitively regulated by various homeostatic responses that typically include effectors of polyamine biosynthesis, catabolism, and transport. Best known among these are the key enzymes involved in polyamine biosynthesis, ornithine decarboxylase (ODC) 1 and S-adenosylmethionine decarboxylase. Increases in ODC are often associated with initiation of normal cell growth and with sustained neoplastic cell growth. Overexpression of ODC activity is a well recognized feature of many cancers, and in at least one tumor type, this increase in activity has been attributed to a point mutation leading to stabilization of the typically labile enzyme protein (1). Such findings form the rationale for certain cancer therapeutic and prevention strategies targeting polyamine metabolism. These efforts rely on inhibitors to block biosynthetic enzyme activity (2-5) or, alternatively, on polyamine analogs to down-regulate enzyme activities (6, 7). In the course of studying the cellular effects of bis-ethylated spermidine and spermine analogs, investigators (8 -10) found that in addition to down-regulating ODC and S-adenosylmethionine decarboxylase, certain analogs potently up-regulate the polyamine catabolic enzyme, spermidine/spermine N 1 -acetyltransferase (SSAT). The enzyme acetylates higher polyamines that are then either excreted out of the cell or acted upon by a flavin-adenine-dependent polyamine oxidase, leading to the back-conversion of spermine (Spm) to spermidine (Spd) and Spd to putrescine (Put) (11,12).In...
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.
We have studied the role of putrescine by using transgenic mouse lines overexpressing the human ornithine decarboxylase gene in most of their tissues. The aberrant expression of the transgene is most strikingly manifested in the brain, leading to an increase of up to 20-fold in putrescine content. We report that the transgenic mice with grossly elevated putrescine in all brain regions analysed (cortex, striatum, hippocampus and cerebellum) showed a significantly elevated seizure threshold to chemical and electrical stimuli, and impaired performance in spatial learning and memory tests. The view that putrescine may be primarily responsible for these changes was supported by the fact that the concentrations of the major neurotransmitter amino acids, glutamate and GABA in the brain, were not changed in the transgenic animals, and by the finding that a further increase in brain putrescine, achieved by inhibition of the catabolism of L-ornithine, appeared to provide additional protection against electroshock-induced seizures. These results suggest that the commonly observed increase in ornithine decarboxylase activity and the massive increase in brain putrescine in connection with neuron damage is a neuroprotective measure rather than a cause of the damage.
The tie gene encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, particularly during embryonic development and angiogenesis in adults. We have cloned and characterized the mouse tie gene and isolated the human and mouse tie promoters. The promoter activities of human and mouse tie were analyzed using luciferase reporter gene constructs in transfected cell lines and beta-galactosidase constructs in transgenic mice. In transfection assays of cultured cells, both human and mouse promoter DNA fragments showed activity that was not restricted to endothelial cells. In contrast, in transgenic mice both promoters directed expression of the reporter gene to endothelial cells undergoing vasculogenesis and angiogenesis. In adult mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries. Our results show that the endothelial cell-type specificity of the tie promoter in vivo can be transferred to heterologous genes by using relatively short promoter fragments. The tie promoter, thus, has useful properties for potential gene therapy.
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