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

Abstract

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...

Activated Polyamine Catabolism Depletes Acetyl-CoA Pools and Suppresses Prostate Tumor Growth in TRAMP Mice

“…The effort was catalyzed by our recent reports showing that conditional overexpression of SSAT inhibits in vitro growth of both MCF-7 breast carcinoma cells (20) and LNCaP prostate carcinoma cells (21). Consistent with these findings, we now demonstrate that overexpression of SSAT markedly suppresses tumor outgrowth of early and advanced prostatic cancer in TRAMP mice.…”

“…We have previously reported that conditional overexpression of SSAT in MCF-7 breast carcinoma cells leads to polyamine pool depletion and growth inhibition (20). As a prelude to the present study, we showed that conditional enzyme overexpression in LNCaP prostate carcinoma cells causes growth inhibition that differed from that seen in MCF-7 cells in that it was not accompanied by polyamine pool depletion (21).…”

Metabolic and Antiproliferative Consequences of Activated Polyamine Catabolism in LNCaP Prostate Carcinoma Cells

“…5). Intracellular AcSpd increased remarkably from undetectable levels (Ͻ10 pmol/10 6 cells) to 10,420 pmol/10 6 cells by 48 h. Other SSAT products, AcSpm and DiAcSpm, which are rarely seen in cells (31), accumulated to 390 pmol/10 6 cells and 2,340 pmol/10 6 cells, respectively, by 48 h and remained elevated during the course of the 144-h experiment. Putrescine (Put) pools also rose remarkably due presumably to back-conversion of Put from Spd via AcSpd (Fig.…”

Peroxisome proliferator‐activated receptor δ as a molecular target to regulate lung cancer cell growth

“…It has been reported that the activation of PPARd stimulates polyamine catabolism via the induction of SSAT, leading to a decreased polyamine level in cancer cells [31] and that indomethacin treatment reduced the polyamine level in tumor tissues [28]. In addition to these reports, the cellular polyamine level is considered a key determinant to induce a cytostatic or cytotoxic effect in cells [32,33]. Overall, it is likely that the cellular polyamine level in A549 cells acts as a determinant to induce cytostatic or cytotoxic effects as observed in this study.…”