Effects of transient expression of spermidine/spermine N 1 ‐acetyltransferase in COS cells

117

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...

Section: Discussionmentioning

confidence: 99%

Effects of Conditional Overexpression of Spermidine/Spermine N 1-Acetyltransferase on Polyamine Pool Dynamics, Cell Growth, and Sensitivity to Polyamine Analogs

Vujcic¹,

Halmekytö²,

Diegelman

et al. 2000

117

“…The gene has been transiently expressed in COS cells where it led to an accumulation of N 1 -acetylspermidine and putrescine, a decrease in spermidine and spermine pools, and a compensatory increase in biosynthetic enzyme activities (20). Possible effects on cell growth were not reported perhaps because of the transient nature of the expressed enzyme activity (20). Some insight into the cellular consequences of SSAT overexpression has been provided by studies with polyamine analogs such as N 1 ,N…”

mentioning

confidence: 99%

“…Attempts to constitutively overexpress SSAT in mammalian cells have been unsuccessful due to poor translation of message in the absence of elevated intracellular polyamines (19). The gene has been transiently expressed in COS cells where it led to an accumulation of N 1 -acetylspermidine and putrescine, a decrease in spermidine and spermine pools, and a compensatory increase in biosynthetic enzyme activities (20). Possible effects on cell growth were not reported perhaps because of the transient nature of the expressed enzyme activity (20).…”

mentioning

confidence: 99%

Activation of Polyamine Catabolism Profoundly Alters Tissue Polyamine Pools and Affects Hair Growth and Female Fertility in Transgenic Mice Overexpressing Spermidine/SpermineN 1-Acetyltransferase

Pietilä¹,

Alhonen²,

Halmekytö³

et al. 1997

111

141

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...

“…Previous attempts to constitutively overexpress the enzyme in mammalian cells have been relatively unsuccessful, due apparently to poor translation of the SSAT message in the absence of elevated levels of intracellular polyamines (13,14). The gene has been transiently expressed in COS cells leading to an accumulation of putrescine and N 1 -acetylspermidine, a decrease in spermidine and spermine pools, and enhanced biosynthetic enzyme activities (15). However, due to the transient nature of the effect, the relationship of increased SSAT gene expression to cell growth was not clearly resolved.…”

mentioning

confidence: 99%

Correlation of Polyamine and Growth Responses to N 1,N 11-Diethylnorspermine in Primary Fetal Fibroblasts Derived from Transgenic Mice Overexpressing Spermidine/SpermineN 1-Acetyltransferase

Alhonen¹,

Karppinen²,

Uusi‐Oukari³

et al. 1998

A recently generated transgenic mouse line having activated polyamine catabolism due to systemic overexpression of spermidine/spermine N 1 -acetyltransferase (SSAT) was used to isolate primary fetal fibroblasts as a means to further elucidate the cellular consequences of activated polyamine catabolism. Basal levels of SSAT activity and steady-state mRNA in the transgenic fibroblasts were about ϳ20-and ϳ40-fold higher than in nontransgenic fibroblasts. Consistent with activated polyamine catabolism, there was an overaccumulation of putrescine and N 1 -acetylspermidine and a decrease in spermidine and spermine pools. Treatment with the polyamine analogue N 1 ,N 11 -diethylnorspermine (DENSPM) increased SSAT activity in the transgenic fibroblasts ϳ380-fold, whereas mRNA increased only ϳ3-fold, indicating post-mRNA regulation. SSAT activity in the nontransgenic fibroblasts increased ϳ200-fold. By Western blot, enzyme protein was found to increase ϳ46 times higher in the treated transgenic fibroblasts than non-transgenic fibroblasts: a value comparable to 36-fold differential in enzyme activity. With DENSPM treatment, spermidine pools were more rapidly depleted in the transgenic fibroblasts than in nontransgenic fibroblasts. Similarly, transgenic fibroblasts were much more sensitive to DENSPM-induced growth inhibition. This was not diminished by co-treatment with an inhibitor of polyamine oxidase, suggesting that growth inhibition was due to polyamine depletion per se as opposed to oxidative stress. Since the two fibroblasts were genetically identical except for the transgene, the various metabolic and growth response differences are directly attributable to overexpression of SSAT.Deregulated polyamine biosynthesis as indicated by overexpressed ornithine decarboxylase (ODC) 1 activity is a well recognized characteristic of animal and human cancers. In at least one tumor type, the genetic basis for this has been attributed to a point mutation leading to stabilization of the enzyme protein (1). Although several groups have reported that ODC may play a causative role in the process of cell transformation in cultured systems (2)(3)(4), that function appears to be attenuated in vivo. Transgenic mice that systemically overexpress native ODC by at least 20-fold fail to show an increased incidence of tumors in any of their tissues (5). More likely, the enzyme interacts with other genes to fulfill a facilitating role in tumorigenesis not unlike that seen with oncogene cooperativity, a phenomenon that has been previously demonstrated between ODC and ras (6). This potential is clearly indicated in mouse skin carcinogenesis models where induction of ODC activity represents an early response to tumor promoters (7) and is known to be critical to tumor formation (8, 9). In this regard, O'Brien's group (10) recently reported that transgenic mice that overexpress a stabilized form of ODC in the skin are much more susceptible to 7,12-dimethylbenz(a)anthracene-induced carcinogenesis. In addition, their findings strongly suggest that am...