Effects of conditional overexpression of spermidine/spermine N1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs

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 approximately 20-fold increase in SSAT RNA and a approximately 10-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 approximately 100-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.     

Involvement of polyamines in evening primrose extract-induced apoptosis in Ehrlich ascites tumor cells

Summary. We previously demonstrated that evening primrose extract (EPE) induced apoptosis and inhibited the DNA synthesis in Ehrlich ascites tumor cells (EATC) and suggested that EPE-induced inhibition of the growth of EATC are via at least two pathway differentially modulated by reactive oxygen species, notably intracellular peroxides. These are (a) the EPE-induced apoptosis pathway which is dependent on increases in hydrogen peroxide and (b) the EPE-induced inhibition of cell proliferation which is hydrogen peroxide independent. In this study, EPE brought about a significant decrease in intracellular polyamine levels. Furthermore, the addition of polyamines reversed the EPE-induced decrease in cell viability and suppressed the EPE-induced increase in intracellular hydrogen peroxides. However, the addition of polyamines did not reverse EPE-induced decrease in DNA synthesis and phosphorylation of Rb protein, and EPE-induced translocation of AIF. These results suggest the involvement of polyamines in the EPE-induced apoptosis pathway which is dependent on increase in hydrogen peroxide.     

Inhibition of spermidine and spermine synthesis leads to growth arrest of rat embryo fibroblasts in G1

Following growth stimulation of rat embryo fibroblast (REF) cells previously arrested in G₁ by serum deprivation, there occurs a large increase in the synthesis of the polyamines putrescine, spermidine and spermine. Methylglyoxal bis(guanylhydrazone) (MGBG), a potent inhibitor of S-adenosylmethionine decarboxylase can block the accumulation of both spermidine and spermine over a period of several days. Under such conditions REF cells treated with MGBG will approximately double in number and then become growth-arrested again predominantly in the G₁ phase of the cell cycle. REF cells therefore appear to contain sufficient spermidine and spermine to progress through one cell cycle before the intracellular levels of these polyamines is reduced sufficiently to arrest growth in the absence of continued polyamine synthesis. Limitation of intracellular polyamine levels is therefore not the mechanism by which deprivation of serum growth factors arrests cell growth. While continued growth is nevertheless dependent on polyamine synthesis, this cell type is capable of limited proliferation in its absence. Addition of spermidine or spermine to MGBG-arrested REF cells results in a rapid resumption of proliferation demonstrating that either polyamine can fulfill the role played by these polyamines in the growth process. Low levels of spermidine and spermine therefore arrest this cell type at a resriction point in G₁ at which it is decided whether the intracellular level of these polyamines is sufficiently high to enable a cell to enter into and complete a new cell cycle. This polyamine-sensitive restriction point is considered to be analogous to the restriction point(s) in G₁ at which serum and nutrient limitation act.     

Metabolic and antiproliferative consequences of activated polyamine catabolism in LNCaP prostate carcinoma cells

Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, we reasoned that this might be more effectively achieved by activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT); a strategy first validated in MCF-7 breast carcinoma cells. We now examine the possibility that, due to unique aspects of polyamine homeostasis in the prostate gland, tumor cells derived from it may be particularly sensitive to activated polyamine catabolism. Thus, SSAT was conditionally overexpressed in LNCaP prostate carcinoma cells via a tetracycline-regulatable (Tet-off) system. Tetracycline removal resulted in a rapid approximately 10-fold increase in SSAT mRNA and an increase of approximately 20-fold in enzyme activity. SSAT products N(1)-acetylspermidine, N(1)-acetylspermine, and N(1),N(12)-diacetylspermine accumulated intracellularly and extracellularly. SSAT induction also led to a growth inhibition that was not accompanied by polyamine pool depletion as it was in MCF-7 cells. Rather, intracellular spermidine and spermine pools were maintained at or above control levels by a robust compensatory increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase activities. This, in turn, gave rise to a high rate of metabolic flux through both the biosynthetic and catabolic arms of polyamine metabolism. Treatment with the biosynthesis inhibitor alpha-difluoromethylornithine during tetracycline removal interrupted flux and prevented growth inhibition. Thus, flux-induced growth inhibition appears to derive from overaccumulation of metabolic products and/or from depletion of metabolic precursors. Metabolic effects that were not excluded as possible contributing factors include high levels of putrescine and acetylated polyamines, a 50% reduction in S-adenosylmethionine, and a 45% decline in the SSAT cofactor acetyl-CoA. Overall, the study demonstrates that activation of polyamine catabolism in LNCaP cells elicits a compensatory increase in polyamine biosynthesis and downstream metabolic events that culminate in growth inhibition.     

Regulation of polyamine transport in Chinese hamster ovary cells

Control Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with alpha-difluoromethylornithine in CHO cells or via polyamine starvation of CHODC- cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.     

Factors affecting polyamine excretion from mammalian cells in culture. Inhibitors of polyamine biosynthesis

Canavanine, diaminopropane, alpha-methylornithine and methylglyoxal bis(guanylhydrazone) decreased the intracellular polyamine concentrations in growing baby hamster kidney cells. Each of the inhibitors also prevented polyamine efflux into the extracellular medium. Concomitant with the decrease in polyamine excretion was a change in the distribution of polyamines in the extracellular medium. In each case there was a decrease in the amount of radioactivity present as free spermidine and an increase in that found as acetyl polyamines. The magnitude of this shift correlated with the degree of inhibition of excretion. It may be that acetyl polyamines play a role in the regulation of polyamine excretion.     

Transglutaminase activity is involved in polyamine-induced programmed cell death.

Natural polyamines, i.e., putrescine, spermidine, and spermine, are ubiquitous molecules essential for cell proliferation and differentiation. In the present study, the effect of polyamines on primary cultures of bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and a human melanoma cell line was examined. While in the absence of fetal calf serum (FCS) polyamines had no effect on viability, in the presence of FCS spermidine and spermine, at concentrations close to physiologic levels, induced a dose-dependent cell death, whereas putrescine was ineffective. RASMCs were significantly more sensitive than other cells. FACS analysis, oligo-nucleosome ELISA, Hoechst nuclear staining, and Annexin V-FITC quantification showed that cell death was likely due to apoptosis. Cells exposed to spermidine showed a marked increase of intracellular transglutaminase (TGase) activity ( approximately 30-fold over control). Inhibitors of polyamine oxidation or inhibitors of TGase activity prevented polyamine-induced apoptosis. Moreover, tissue TGase overexpression significantly increased cell sensitivity to polyamine, suggesting that this effect is likely related to enhanced intracellular TGase activity. These data indicate that polyamines may modulate cell viability through a novel TGase-dependent process.     

Diethylglyoxal bis(guanylhydrazone), a potent inhibitor of mammalian S-adenosylmethionine decarboxylase. Effects on cell proliferation and polyamine metabolism in L1210 leukemia cells

The polyamines are cell constituents essential for growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in the polyamine biosynthetic pathway. Methylglyoxal bis(guanylhydrazone) (MGBG) is an anti-leukemic agent with a strong inhibitory effect against AdoMetDC. However, the lack of specificity limits the usefulness of MGBG. In the present report we have used an analog of MGBG, diethylglyoxal bis(guanylhydrazone) (DEGBG), with a much greater specificity and potency against AdoMetDC, to investigate the effects of AdoMetDC inhibition on cell proliferation and polyamine metabolism in mouse L1210 leukemia cells. DEGBG was shown to effectively inhibit AdoMetDC activity in exponentially growing L1210 cells. The inhibition of AdoMetDC was reflected in a marked decrease in the cellular concentrations of spermidine and spermine. The concentration of putrescine, on the other hand, was greatly increased. Treatment with DEGBG resulted in a compensatory increase in the synthesis of AdoMetDC demonstrating an efficient feedback control. Cells seeded in the presece of DEGBG ceased to grow after a lag period of 1–2 days, indicating that the cells contained an excess of polyamines which were sufficient for one or two cell cycles in the absence of polyamine synthesis. The present results indicate that analogs of MGBG, having a greater specificity against AdoMetDC, might be valuable for studies concerning polyamines and cell proliferation.     

Isolation of a polyamine transport deficient cell line from the human non-small cell lung carcinoma line NCI H157

In an effort to study the mechanism underlying the observed phenotype-specific response of human lung cancer cell lines to a polyamine analogue, N¹,N¹²-bis(ethyl)spermine(BESpm), we have isolated a BESpm resistant cell line from the BESpm-sensitive large cell lung carcinoma line NCIH157. The mutant line exhibits identical growth rates in the presence or absence of the analogue. However, the overall growth of mutant cells reaches stationary phase earlier than that of the parental cells. In contrast to the parental cells, where a superinduction of spermidine/spermine N¹-acetyltransferase (SSAT) is associated with BESpm toxicity, treatment of this resistant line with BESpm did not induce SSAT mRNA or enzyme activity. BESpm treatment was not effective in depleting the intracellular polyamine pools and very low intracellular BESpm levels were detected. This BESpm resistance is not mediated by multidrug resistance (MDR) protein, since these cells maintain their sensitivity to the antineoplastic agent adriamycin. Treatment of these cells with methylglyoxal bis(guanylhydrazone) (MGBG), an AdoMetDC inhibitor which enters cell using polyamine transport system, shows no inhibition of cell growth. Our data suggest that these mutant cells are deficient in polyamine transport. Consistent with this hypothesis, exogenous polyamines did not prevent difluoromethylomithine (DFMO) induced growth inhibition in the mutant cells. © 1996 Wiley-Liss, Inc.     

Role of polyamines in gibberellin-induced internode growth in peas

To determine the requirement for polyamines in gibberellin (GA) induced internode growth polyamine content was measured in internodes of peas of various internode phenotypes (slender, tall, dwarf, nana) with and without applied gibberellin (GA₃) and polyamine synthesis inhibitors. Polyamines were assayed as dansyl derivatives which were separated by reverse phase high performance liquid chromatography and detected by fluorescence spectrophotometry. The amounts of polyamines in the different genetic lines of peas, which differed in internode lengths and extractable GA content, correlated with the extent of internode elongation. High polyamine concentrations were associated with young internodes and decreased with internode expansion. Extremely short internodes of nana plants without GA exhibited equal or higher amine concentrations relative to internodes of other lines of peas and GA-stimulated nana seedlings. The polyamine synthesis inhibitors, α-difluoromethylornithine and α-difluoromethylarginine, independently or in combination, inhibited polyamine accumulation and internode elongation of tall peas and GA-stimulated nana plants. Agmatine and putrescine restored growth and endogenous polyamine content to variable degrees. However, exogenous polyamines were not effective in promoting growth unless intracellular amines were partially depleted.

These results suggest that polyamines do not have a role in cell elongation, but may be required to support cell proliferation. Polyamines do not mediate the entire action of GA in internode growth of peas since GA induction of growth involves both cell division and cell elongation, whereas polyamines appear to affect cell division only.

     

DNA binding activity of casein kinase II

Casein kinase II, an ubiquitous, oligomeric, messenger-independent protein kinase has previously been shown to concentrate in the nuclear compartment when cells are stimulated to proliferate. The present communication reports that purified mammalian CKII interacts with genomic DNA preparations in vitro. This interaction led to an apparent activation of the kinase, most likely explained by prevention of its aggregation and subsequent denaturation. Binding of CKII was optimum with double stranded DNA preparations; duplex lambda phage DNA exhibited at least two types of binding sites and the high affinity system (Kd approximately equal to 6 x 10(-13) M) represented a binding capacity of about 1 mol CKII per mol DNA. CKII-DNA interaction was stimulated in the presence of a polyamine and inhibited by heparin. Blotting experiments disclosed that DNA binds CKII through its alpha subunit. These observations are in line with the hypothesis that casein kinase II may be examined as a component in the transduction of the mitogenic signal from the cell membrane to the nucleus, in response to growth factors.     

Differential effects of lysosomotropic amines and polyamines on processing and biological activity of EGF

The effects of lysosomotropic amines and polyamines on rat fibroblasts were studied after the administration of epidermal growth factor (EGF) in order to determine whether the intracellular processing of EGF was important for transmission of its biological signal. Following the addition of EGF, cell cultures exhibited a dose-dependent increase in ornithine decarboxylase (ODC) activity. This increase in ODC activity was drastically reduced by both methylamine, a representative lysosomotropic amine, and putrescine, a polyamine precursor. However, inasmuch as methylamine inhibited EGF-induced DNA synthesis by greater than 50%, putrescine had no inhibitory effect. Lysosomotropic amines, but not polyamines, prevented EGF processing as evidenced by their ability to block the release of intracellular 125EGF and by their ability to inhibit the formation of the final intracellular processed product of EGF, as determined by isoelectric focusing. These data suggest that the processing of EGF is consistent with the induction of DNA synthesis and ODC activity. The cellular mechanisms involved in inhibition of ODC induction by polyamines appear to be distinct from those involved in lysosomotropic amines.     

Lack of a correlation between polyamine synthesis and DNA synthesis by cultured rat liver cells and fibroblasts

Growth stimulation of either fetal rat liver cells or rat embryo fibroblasts in culture results in considerable increases in intracellular polyamine levels as cells proceed through the cell cycle. Treatment of such cell cultures with appropriate levels of two inhibitors of polyamine synthesis, namely α-hydrazino ornithine and methylglyoxal bis(guanylhydrazone), can essentially completely block these increases in cellular polyamine content. Under such conditions, where the elevation in intracellular polyamine content is prevented, cell cultures are nevertheless able to initiate DNA synthesis and subsequently synthesize DNA at rates comparable to untreated control cultures that have been growth-stimulated. These two cell types therefore contain sufficient polyamines when in a resting state (G₁) to enable them to enter from G₁ into S phase and traverse S phase at normal rates in the absence of further polyamine synthesis. The recruitment of cells into the first cell cycle, through serum stimulation of growth, therefore appears not to be mediated or regulated by the increases in intracellular levels of polyamines that occurs under these conditions. Conversely, the arrest of growth of these cell types resulting from serum deprivation is not mediated by a limitation of intracellular polyamine content.     

Uptake and excretion of polyamines from baby hamster kidney cells (BHK-21/C13) the effect of serum on confluent cell cultures

In confluent cultures of BHK-21/C13 cells there was little uptake oxogenous polyamines and only a low level of polyamine biosynthesis. These cultures continously excreted polyamines into the extracellular medium. Spermidine, in both the free and bound form, was the predominant excretion product, whereas the major intracellular polyamine was spermine implying that excretion of polyamines was specific. Reinitiation of growth by the addition of fresh serum immediately increased the uptake of exogenous putrescine, increased the biosynthesis of polyamines and decreased the excretion of polyamines. Thus, polyamine transport into and out of the cell appears to be regulated by the growth status of that cell.     

Novel lysine-spermine conjugate inhibits polyamine transport and inhibits cell growth when given with DFMO

Polyamines are ubiquitous molecules with multiple intracellular functions. Cells tightly regulate their levels through feedback mechanisms affecting synthesis, intracellular conversion, and transport. Because polyamines have an important role in regulating cell growth, they are a target for cancer therapeutic development. However, to effectively inhibit cell growth through polyamine depletion one needs to inhibit both polyamine synthesis and import. Although the mammalian polyamine transporter has not been cloned, we have identified ORI 1202, an N(1)-spermine-L-lysinyl amide, as an effective polyamine transport inhibitor. ORI 1202 prevents the cellular accumulation of [(3)H]spermidine over a 20-h test period. ORI 1202 (30-100 microM) effectively inhibits cell growth when used in conjunction with the polyamine synthesis inhibitor alpha-difluoromethylornithine (DFMO; > or =230 microM). Human breast, prostate, and bladder carcinoma cell lines and melanoma cell lines show ORI 1202 EC(50) values in the low micromolar range when tested in conjunction with DFMO. This cytostatic effect correlates with a reduction in the intracellular levels of putrescine and spermidine. When ORI 1202 (45 mg/kg, i.p., tidx5) and DFMO (1% in drinking water) were delivered over 14 days, MDA-MB-231 breast tumor xenografts in nude mice showed 50% growth inhibition. Polyamine depletion therapy provides a cytostatic therapy that could be useful against cancer and other diseases resulting from uncontrolled cell growth.     

Polyamine acetylation modulates polyamine metabolic flux, a prelude to broader metabolic consequences

Recent studies suggest that overexpression of the polyamine-acetylating enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) significantly increases metabolic flux through the polyamine pathway. The concept derives from the observation that SSAT-induced acetylation of polyamines gives rise to a compensatory increase in biosynthesis and presumably to increased flow through the pathway. Despite the strength of this deduction, the existence of heightened polyamine flux has not yet been experimentally demonstrated. Here, we use the artificial polyamine precursor 4-fluoro-ornithine to measure polyamine flux by tracking fluorine unit permeation of polyamine pools in human prostate carcinoma LNCaP cells. Conditional overexpression of SSAT was accompanied by a massive increase in intracellular and extracellular acetylated spermidine and by a 6-20-fold increase in biosynthetic enzyme activities. In the presence of 300 microM 4-fluoro-ornithine, SSAT overexpression led to the sequential appearance of fluorinated putrescine, spermidine, acetylated spermidine, and spermine. As fluorinated polyamines increased, endogenous polyamines decreased, so that the total polyamine pool size remained relatively constant. At 24 h, 56% of the spermine pool in the induced SSAT cells was fluorine-labeled compared with only 12% in uninduced cells. Thus, SSAT induction increased metabolic flux by approximately 5-fold. Flux could be interrupted by inhibition of polyamine biosynthesis but not by inhibition of polyamine oxidation. Overall, the findings are consistent with a paradigm whereby flux is initiated by SSAT acetylation of spermine and particularly spermidine followed by a marked increase in key biosynthetic enzymes. The latter sustains the flux cycle by providing a constant supply of polyamines for subsequent acetylation by SSAT. The broader metabolic implications of this futile metabolic cycling are discussed in detail.     

Polyamine depletion induces apoptosis through mitochondria-mediated pathway

Polyamines, namely putrescine, spermidine, and spermine, are essential for cell survival and proliferation. A decrease in intracellular polyamine levels is associated with apoptosis. In this study, we used inhibitors of polyamine biosynthesis to examine the effect of polyamine depletion. A combination of inhibitors of ornithine decarboxylase, S-adenosylmethionine decarboxylase, or spermidine synthase decreased intracellular polyamine levels and induced cell death in a WEHI231 murine B cell line. These cells exhibited apoptotic features including chromatin condensation and oligonucleosomal DNA fragmentation. Addition of exogenous polyamines reversed the observed features of apoptotic cell death. Similar effects were also observed in other cell lines: a human B cell line Ramos and a human T cell line Jurkat. Depletion of polyamines induced activation of caspase-3 and disruption of the mitochondrial membrane potential (Delta psi m). Inhibition of caspase activities by an inhibitor prevented the apoptotic nuclear changes but not Delta psi m disruption induced by polyamine depletion. Overexpression of Bcl-xl, an anti-apoptotic Bcl-2 family protein, completely inhibited Delta psi m disruption, caspase activation, and cell death. These results indicate that the depletion of intracellular polyamines triggers the mitochondria-mediated pathway for apoptosis, resulting in caspase activation and apoptotic cell death.