Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues

We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.     

Subcellular distribution of spermidine/spermine N1-acetyltransferase

The subcellular distribution of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) was studied in L56Br-C1 cells treated with 10 microM N(1),N(11)-diethylnorspermine (DENSPM) for 24 h. Cells were fractioned into three subcellular fractions. A particulate fraction containing the mitochondria was denoted as the mitochondrial fraction. After DENSPM treatment, an increase in SSAT activity was mainly found in the mitochondrial fraction. Western blot analysis showed an increased level of the SSAT protein in the mitochondrial fraction compared to the cytosolic fraction. Immunofluorescence microscopy and immunogold labeling transmission electron microscopy also showed a mitochondrial association of SSAT. Transmission electron microscopy revealed that the endoplasmic reticulum was devoid of ribosomes in DENSPM-treated cells, in contrast to control cells that contained ample ribosomes. An increased SSAT activity in connection with the mitochondria may be part of the mechanism of DENSPM-induced apoptosis.     

Tissue-specific alternative splicing of spermidine/spermine N 1-acetyltransferase

The polyamines, spermidine and spermine, are abundant organic cations participating in many important cellular processes. We have previously shown that the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N ¹-acetyltransferase (SSAT), has an alternative mRNA splice variant (SSATX) which undergoes degradation via nonsense-mediated mRNA decay (NMD) pathway, and that the intracellular polyamine level regulates the ratio of the SSATX and SSAT splice variants. The aim of this study was to investigate the effect of SSATX level manipulation on SSAT activity in cell culture, and to examine the in vivo expression levels of SSATX and SSAT mRNA. Silencing SSATX expression with small interfering RNA led to increased SSAT activity. Furthermore, transfection of SSAT-deficient cells with mutated SSAT gene (which produced only trace amount of SSATX) yielded higher SSAT activity than transfection with natural SSAT gene (which produced both SSAT and SSATX). Blocking NMD in vivo by protein synthesis inhibitor cycloheximide resulted in accumulation of SSATX mRNA, and like in cell culture, the increase of SSATX mRNA was prevented by administration of polyamine analog N ¹ ,N ¹¹ -diethylnorspermine. Although SSATX/total SSAT mRNA ratio did not correlate with polyamine levels or SSAT activity between different tissues, increasing polyamine levels in a given tissue led to decreased SSATX/total SSAT mRNA ratio and vice versa. Taken together, the regulated unproductive splicing and translation of SSAT has a physiological relevance in modulating SSAT activity. However, in addition to polyamine level there seems to be additional factors regulating tissue-specific alternative splicing of SSAT.     

Mechanistic and structural analysis of human spermidine/spermine N1-acetyltransferase

The N1-acetylation of spermidine and spermine by spermidine/spermine acetyltransferase (SSAT) is a crucial step in the regulation of the cellular polyamine levels in eukaryotic cells. Altered polyamine levels are associated with a variety of cancers as well as other diseases, and key enzymes in the polyamine pathway, including SSAT, are being explored as potential therapeutic drug targets. We have expressed and purified human SSAT in Escherichia coli and characterized its kinetic and chemical mechanism. Initial velocity and inhibition studies support a random sequential mechanism for the enzyme. The bisubstrate analogue, N1-spermine-acetyl-coenzyme A, exhibited linear, competitive inhibition against both substrates with a true Ki of 6 nM. The pH-activity profile was bell-shaped, depending on the ionization state of two groups exhibiting apparent pKa values of 7.27 and 8.87. The three-dimensional crystal structure of SSAT with bound bisubstrate inhibitor was determined at 2.3 A resolution. The structure of the SSAT-spermine-acetyl-coenzyme A complex suggested that Tyr140 acts as general acid and Glu92, through one or more water molecules, acts as the general base during catalysis. On the basis of kinetic properties, pH dependence, and structural information, we propose an acid/base-assisted reaction catalyzed by SSAT, involving a ternary complex.     

Significant induction of spermidine/spermine N1-acetyltransferase without cytotoxicity by the growth-supporting polyamine analogue 1,12-dimethylspermine

The superinduction of the polyamine catabolic enzyme spermidine/spermine N¹-acetyltransferase (SSAT) has been implicated in the cell type-specific cytotoxic activity of some polyamine analogues. We now report that one polyamine analogue, 1, 12-dimethylspermine (DMSpm), produces a large induction of SSAT with no significant effects on growth in the human large cell lung carcinoma line, NCI H157. This cell line has been demonstrated to respond to other analogues with SSAT superinduction and cell death. Treatment of the lung cancer cell line with DMSpm produces a rapid increase in SSAT activity and a near complete depletion of the natural polyamines. Additionally, DMSpm supports cell growth in cells which have been depleted of their natural polyamines by the ornithine decarboxylase inhibitor, 2-difluoromethylornithine. The current results suggest that significant induction of SSAT can occur in the absence of cytotoxicity when the inducing polyamine analogue can support growth and that increased SSAT activity alone is not sufficient for cytotoxicity to occur. © 1995 Wiley-Liss Inc.     

Properties of the spermidine/spermine N1-acetyltransferase mutant L156F that decreases cellular sensitivity to the polyamine analogue N1, N11-bis(ethyl)norspermine

Properties of a mutant form of spermidine/spermine N(1)-acetyltransferase, L156F (L156F-SSAT), that is present in Chinese hamster ovary cells selected for resistance to the polyamine analogue N(1,) N(11)-bis(ethyl)norspermine (BE 3-3-3) were investigated. Increased K(m) values, decreased V(max) values, and decreased k(cat) values with both polyamine substrates, spermidine and spermine, indicated that L156F-SSAT is an inferior and less efficient acetyltransferase than wild-type SSAT. Transfection of L156F-SSAT into C55.7Res cells indicated that cellular SSAT activity per nanogram of SSAT protein correlated well with the in vitro data and was also approximately 20-fold less for the mutant protein than for wild-type SSAT. Increased expression of L156F-SSAT was unable to restore cellular sensitivity to BE 3-3-3 despite providing measurable basal SSAT activity. Only a 4-fold induction of L156F-SSAT activity resulted from the exposure of cells to the polyamine analogue, whereas wild-type SSAT was induced approximately 300-fold. Degradation studies indicated that BE 3-3-3 cannot prevent ubiquitination of L156F-SSAT and is therefore unable to protect the mutant protein from degradation. These studies indicate that the decreased cellular sensitivity to BE 3-3-3 is caused by the lack of SSAT activity induction in the presence of the analogue due to its inability to prevent the rapid degradation of the L156F-SSAT protein.     

Altered spermidine/spermine N1-acetyltransferase activity as a mechanism of cellular resistance to bis(ethyl)polyamine analogues

To develop a model system to investigate mechanisms of antiproliferative action of bis(ethyl)polyamine analogues, intermittent analogue treatments followed by recovery periods in drug-free medium were used to select an N(1), N(12)-bis(ethyl)spermine-resistant derivative of the Chinese hamster ovary cell line C55.7. The resulting C55.7Res line was at least 10-fold resistant to N(1),N(12)-bis(ethyl)spermine and N(1), N(11)-bis(ethyl)norspermine. The stability of the resistance in the absence of selection pressure was >/=9 months, indicating that a heritable genotypic change was responsible for the resistance phenotype. Polyamine transport alterations and multi-drug resistance were eliminated as causes of the resistance. Spermidine/spermine N(1)-acetyltransferase (SSAT) activity and regulation were altered in C55.7Res cells as basal activity was decreased, and no activity induction resulted from exposure to analogue concentrations, which caused 300-fold enzyme induction in parental cells. SSAT mRNA levels in the absence and presence of analogue were unchanged, but no SSAT protein was detected in C55.7Res cells. A point mutation, which results in the change leucine156 (a fully conserved residue) to phenylalanine, was identified in the C55.7Res SSAT cDNA. Expression of wtSSAT activity in C55.7Res cells restored sensitivity to bis(ethyl)polyamines. These results provided definitive evidence that SSAT activity is a critical target of the cytotoxic action of these analogues.     

Characterization of human spermidine/spermine N1-acetyltransferase purified from cultured melanoma cells

Extreme inducibility of spermidine/spermine acetyltransferase (SSAT) by bis-ethyl derivatives of spermine in human large cell lung carcinoma and melanoma cells has prompted biochemical characterization of the purified enzyme. Treatment of human MALME-3 melanoma cells with 10 microM N1,N11-bis(ethyl)norspermine (BENSPM) for 48-72 h increased SSAT activity by some 1000- to 4000-fold and enabled purification of the enzyme by established procedures--binding on immobilized spermine and elution with spermine followed by binding on Matrex Blue A and elution with coenzyme A. The enzyme showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a single subunit species and molecular weight of approximately 20,300 Da. By gel permeation chromatography, the holoenzyme was found to have a molecular weight of 80,000 Da, suggesting a total of four identical subunits. Purified SSAT had a specific activity of 285 mumol/min/mg for spermidine and Km values of 5.9 microM for acetylcoenzyme A, 55 microM for spermidine, 5 microM for spermine, 36 microM for N1-acetylspermine, 1.6 microM for norspermidine, and 4 microM for norspermine. Homologs of BENSPM were found to be competitive inhibitors of spermidine acetylation, with Ki values of 0.8 microM for BENSPM, 1.9 microM for N1,N12-bis-(ethyl)spermine and 17 microM for N1,N14-bis-(ethyl)-homospermine. Correlation of these values with the relative abilities of the homologs to increase SSAT in intact cells suggests that formation of an enzyme inhibitor complex may play a contributing role in enzyme induction.     

Induction of spermidine/spermine N1-acetyltransferase by methylglyoxal bis(guanylhydrazone)

The anti-tumor agent methylglyoxal bis(guanylhydrazone) was found to be a competitive inhibitor of spermidine/spermine N1-acetyltransferase with a Ki of about 8 microM. Treatment of rats with this drug lead to a very large increase in the total amount of spermidine/spermine N1-acetyltransferase in liver, kidney and spleen. The total increase as measured using a specific antiserum amounted to 700-fold in liver and 100-fold in kidney within 18 h of treatment with 80 mg/kg doses. At least part of this induction was due to a pronounced increase in the half-life of the acetyltransferase which increased from 15 min to more than 12 h. The very large increase in the amount of the enzyme is likely to overwhelm the direct inhibition, and a net increase in the acetylation of polyamines by this enzyme would be expected to occur after treatment with methylglyoxal bis(guanylhydrazone). The acetylated polyamines are known to be rapidly degraded by polyamine oxidase producing putrescine. Direct evidence that a substantial part of the increase in the content of putrescine in the liver of rats treated with methylglyoxal bis(guanylhydrazone) occurs via the induction of this acetylase/oxidase pathway was obtained. These results indicate that methylglyoxal bis(guanylhydrazone) affects cellular polyamine levels not only by means of its inhibitory effect on S-adenosylmethionine decarboxylase and diamine oxidase but also by the induction of spermidine/spermine N1-acetyltransferase. They also raise the possibility that the enormous increase in this enzyme which occurs with higher doses may contribute to the very severe toxicity of methylglyoxal bis(guanylhydrazone).     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.     

Phorbol esters stimulate spermidine/spermine N1-acetyltransferase activity in mitogen-stimulated bovine lymphocytes

Phorbol 12-myristate-13-acetate (PMA) is shown to induce spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, in bovine lymphocytes. When PMA and phytohemagglutinin (PHA) were added simultaneously, the enzyme activity was stimulated synergistically. The ability of phorbol esters to stimulate the enzyme activity was consistent with their tumor-promoting ability. Phorbol, which is not a tumor promotor, was incapable of stimulating the enzyme activity. Phorbol diacetate weakly stimulated the activity of the acetylase. Phorbol dibutyrate had a similar stimulatory effect to PMA. These results suggest that the spermidine/spermine N1-acetyltransferase may play an important role in changes in polyamine levels in phorbol ester-treated cells and that the increase in the enzyme activity may have some relationship to the control of cell growth and differentiation by phorbol esters.     

High specific induction of spermidine/spermine N1-acetyltransferase in a human large cell lung carcinoma.

The effects of insulin and anti-(insulin receptor) monoclonal antibodies on tyrosine phosphorylation were investigated in fibroblasts transfected with human insulin receptor cDNA (NIH 3T3HIR3.5 cells) using anti-phosphotyrosine immunoblotting. Insulin increased levels of tyrosine phosphorylation in two major proteins of molecular mass 97 kDa (pp97, assumed to be the insulin receptor beta-subunit) and 185 kDa (pp185). Insulin-mimetic anti-receptor antibodies also stimulated tyrosine phosphorylation of both pp97 and pp185. The observation of antibody-stimulated pp97 phosphorylation, as detected by immunoblotting, is in contrast with previous data which failed to show receptor autophosphorylation in NIH 3T3HIR3.5 cells labelled with [32P]P1. The effect of insulin on pp97 was maximal within 1 min, but the response to antibody was apparent only after a lag of 1-2 min and rose steadily over 20 min. The absolute level of antibody-stimulated phosphorylation of both pp97 and pp185 after 20 min was only about 20% of the maximum level induced by equivalent concentrations of insulin, even at concentrations of antibody sufficient for full occupancy of receptors. Another insulin-mimetic agent, wheat-germ agglutinin, stimulated receptor autophosphorylation with kinetics similar to those produced by the antibody. It is suggested that the relatively slow responses to both agents may be a function of the dependence on receptor cross-linking. These data are consistent with a role for the insulin receptor tyrosine kinase activity in the mechanism of action of insulin-mimetic anti-receptor antibodies.     

Spermidine/spermine N1-acetyltransferase overexpression in kidney epithelial cells disrupts polyamine homeostasis, leads to DNA damage, and causes G2 arrest

Expression of spermidine/spermine N¹-acetyltransferase (SSAT) increases in kidneys subjected to ischemia-reperfusion injury (IRI). Increased expression of SSAT in vitro leads to alterations in cellular polyamine content, depletion of cofactors and precursors of polyamine synthesis, and reduced cell proliferation. In our model system, a >28-fold increase in SSAT levels in HEK-293 cells leads to depletion of polyamines and elevation in the enzymatic activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, suggestive of a compensatory reaction to increased polyamine catabolism. Increased expression of SSAT also led to DNA damage and G₂ arrest. The increased DNA damage was primarily due to the depletion of polyamines. Other factors such as increased production of H₂O₂ due to polyamine oxidase activity may play a secondary role in the induction of DNA lesions. In response to DNA damage the ATM/ATR Chk1/2 DNA repair and cell cycle checkpoint pathways were activated, mediating the G₂ arrest in SSAT-expressing cells. In addition, the activation of ERK1 and ERK2, which play integral roles in the G₂/M transition, is impaired in cells expressing SSAT. These results indicate that the disruption of polyamine homeostasis due to enhanced SSAT activity leads to DNA damage and reduced cell proliferation via activation of DNA repair and cell cycle checkpoint and disruption of Raf MEK ERK pathways. We propose that in kidneys subjected to IRI, one mechanism through which increased expression of SSAT may cause cellular injury and organ damage is through induction of DNA damage and the disruption of cell cycle. ischemia-reperfusion injury; polyamine depletion; cell proliferation; DNA repair; cell cycle arrest     

精脒／精胺N^1-乙酰转移酶与肿瘤

多胺是人体中一类重要的含高密度正电荷的小分子有机化合物,肿瘤细胞快速生长对多胺的依赖使多胺代谢途径成为抗肿瘤治疗的新靶点。精脒／精胺N^1-乙酰转移酶（spermidine／spermine N^1-acetyltransferase,ssAT）是多胺降解代谢的限速酶,在维持细胞内多胺稳恒中发挥重要作用。研究发现,SSAT持续性高表达能促进肿瘤发生,而急性诱导性ssAT高表达则能抑制肿瘤生长和诱导肿瘤细胞凋亡。本文对近年来SSAT与肿瘤相关性最新研究进展进行简要综述。     

Role of protein kinase C in phytohemagglutinin-stimulated induction of spermidine/spermine N1-acetyltransferase

We studied the involvement of protein kinase C in the induction of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine degradation, in bovine lymphocytes. When phytohemagglutinin (PHA) and H-7, a protein kinase inhibitor, were added simultaneously to lymphocyte cultures, the elevation caused by PHA of spermidine/spermine N1-acetyltransferase activity at 24 h after administration was reduced. In cells treated with a lower concentration of PHA, the acetyltransferase activity was enhanced with 12-o-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, and reached the level of cells with a higher concentration of PHA. PHA did not cause maximum induction of the enzyme in cells treated with 160 ng/ml TPA. The induction of this acetyltransferase with PHA is probably mediated by protein kinase C.     

Studies of the induction of spermidine/spermine N1-acetyltransferase using a specific antiserum

A specific antiserum to rat liver spermidine/spermine N1-acetyltransferase was used to study the induction of this protein. The antiserum had no effect on the spermidine acetylating capacity of crude nuclear extracts and very little effect on the activity present in crude cytosolic extracts from control rat tissues indicating that most of this activity is not due to spermidine/spermine N1-acetyltransferase. Treatment of rats with carbon tetrachloride, spermidine, thioacetamide, or methylglyoxal bis(guanylhydrazone) produced a substantial increase in the spermidine acetylating capacity of rat liver cytosolic extracts which was exclusively due to an increase in the immunoprecipitable spermidine/spermine N1-acetyltransferase protein. Exact measurement of the extent of this increase was not possible because the basal amount was too low to determine precisely but the amount of this enzyme increased about 250-fold with 6 h of treatment with carbon tetrachloride, about 25-fold at 6 h after spermidine, about 23-fold at 24 h after thioacetamide and up to 300-fold at 24 h after methylglyoxal bis(guanylhydrazone). Treatment of rats with spermidine also increased spermidine/spermine N1-acetyltransferase in other tissues including lung, kidney, and pancreas. The spermidine/spermine N1-acetyltransferase protein was found to turn over very rapidly with a half-life of about 15 min in thioacetamide-treated rats and 180 min after carbon tetrachloride.     

Studies of the acetyl-CoA-binding site of rat liver spermidine/spermine N1-acetyltransferase.

Rat liver spermidine/spermine N1-acetyltransferase was found to be strongly inhibited by the dyes Cibacron F3GA, Coomassie Brilliant Blue and Congo Red. Inhibition was competitive with respect to acetyl-CoA and Ki values of 0.7 microM and 52 microM were determined for Cibacron F3GA and Coomassie Brilliant Blue respectively. The enzyme was strongly retained by columns of Affi-Gel Blue, which contains Cibacron F3GA linked to agarose. It was not eluted from this adsorbent in the presence of 10 mM-spermidine/0.5 M-NaCl/50 mM-Tris/HCl, pH 7.5, but was released by 1 mM-CoA in 10 mM-spermidine/50 mM-Tris/HCl, pH 7.5. These results are consistent with the presence in the enzyme of a dinucleotide fold that binds acetyl CoA and has a high affinity for Cibacron F3GA. The spermidine/spermine N1-acetyltransferase was irreversibly inactivated by exposure to butane-2,3-dione in sodium borate, pH 7.8, or by exposure to phenylglyoxal or camphorquinone-10-sulphonic acid. All of these reagents are known to interact with arginine residues in proteins under the conditions in which they inactivated the acetyltransferase. Inactivation was prevented by the presence of acetyl-CoA or CoA, but to a lesser extent by 3'-dephospho-CoA and not at all by NAD or adenosine. This protection suggests that an arginine residue at the active site is involved in the binding of the acetyl-CoA substrate. Treatment of the assay mixture but not the spermidine N1-acetyltransferase with alkaline phosphatase prevented the reaction taking place. This suggests that the apparent loss of enzyme activity in response to alkaline phosphatase reported by Matsui, Otani, Kamei & Morisawa [(1982) FEBS Lett. 150, 211-213] is due to dephosphorylation of the acetyl-CoA substrate and that the 3'-phosphate group is essential for activity.