Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, ofS-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.     

Effects of S-adenosyl-1,8-diamino-3-thio-octane and S-methyl-5''-methylthioadenosine on polyamine synthesis in Ehrlich ascites-tumour cells.

The rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are negatively regulated by the polyamines spermidine and spermine. In the present work the spermidine synthase inhibitor S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and the spermine synthase inhibitor S-methyl-5'-methylthioadenosine (MMTA) were used to evaluate the regulatory role of the individual polyamines. Treatment of Ehrlich ascites-tumour cells with AdoDATO caused a marked decrease in spermidine content together with an accumulation of putrescine and spermine. Treatment with MMTA, on the other hand, gave rise to a marked decrease in spermine, with a simultaneous accumulation of spermidine. A dramatic increase in the activity of AdoMetDC, but not of ODC, was observed in MMTA-treated cells. This increase appears to be unrelated to the decrease in spermine content, because a similar rise in AdoMetDC activity was obtained when AdoDATO was given in addition to MMTA, in which case the spermine content remained largely unchanged. Instead, we show that the increase in AdoMetDC activity is mainly due to stabilization of the enzyme, probably by binding of MMTA. Treatment with AdoDATO had no effects on the activities of ODC and AdoMetDC, even though it caused a precipitous decrease in spermidine content. The expected decrease in spermidine-mediated suppression of ODC and AdoMetDC was most probably counteracted by the simultaneous increase in spermine. The combination of AdoDATO and MMTA caused a transient rise in ODC activity. Concomitant with this rise, the putrescine and spermidine contents increased, whereas that of spermine remained virtually unchanged. The increase in ODC activity was due to increased synthesis of the enzyme. There were no major effects on the amount of AdoMetDC mRNA by treatment with the inhibitors, alone or in combination. However, the synthesis of AdoMetDC was slightly stimulated in cells treated with MMTA or AdoDATO plus MMTA. The present study demonstrates that regulation of neither ODC nor AdoMetDC is a direct function of the polyamine structure. Instead, it appears that the biosynthesis of the polyamines is feedback-regulated by the various polyamines at many different levels.     

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spormine

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [³⁵S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescien, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.     

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spermine

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [35S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescine, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.     

Effect of inhibitors of S-adenosylmethionine decarboxylase on the contents of ornithine decarboxylase and S-adenosylmethionine decarboxylase in L1210 cells.

Treatment of L1210 cells with either of two inhibitors of S-adenosylmethionine decarboxylase (AdoMetDC), namely 5'-deoxy-5'-[N-methyl-N-[2-(amino-oxy)ethyl])aminoadenosine or 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine, produced a large increase in the amount of ornithine decarboxylase (ODC) protein. The increased enzyme content was due to a decreased rate of degradation of the protein and to an increased rate of synthesis, but there was no change in its mRNA content. The inhibitors led to a substantial decline in the amounts of intracellular spermidine and spermine, but to a big increase in the amount of putrescine. These results indicate that the content of ODC is negatively regulated by spermidine and spermine at the levels of protein translation and turnover, but that putrescine is much less effective in bringing about this repression. Addition of either spermidine or spermine to the cells treated with the AdoMetDC inhibitors led to a decrease in ODC activity, indicating that either polyamine can bring about this effect, but spermidine produced effects at concentrations similar to those found in the control cells and appears to be the physiologically important regulator. The content of AdoMetDC protein (measured by radioimmunoassay) was also increased by these inhibitors, and a small increase in its mRNA content was observed, but this was insufficient to account for the increase in protein. A substantial stabilization of AdoMetDC occurred in these cells, contributing to the increased enzyme content, but an increase in the rate of translation cannot be ruled out.     

Changes in polyamine metabolism during glucocorticoid-induced programmed cell death in mouse thymus

When mice are injected with dexamethasone, cortical thymocytes are deleted through programmed cell death (PCD). We have used this in vivo model system to investigate the kinetics of PCD and cell proliferation in relation to polyamine metabolism for 16 h after injection of dexamethasone. As a marker for PCD, we used the appearance of a sub-G(1)peak in the DNA histogram. When a sub-G(1)peak appeared at 4 h after dexamethasone treatment, the activity of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) was significantly increased and the activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC) was significantly decreased compared to the activities found in the thymi of control mice. Despite the significant changes in the activities of SSAT and AdoMetDC, the only change in the polyamine pool during the experimental period was that of putrescine. Presumably the complexity of this in vivo system masks changes in the spermidine and spermine pools that were expected in relation to the increased SSAT activity and decreased AdoMetDC activity.     

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.     

Polyamines in testosterone-induced hypertrophic and antifolate-induced hyperplastic mouse kidney. Differential effect of α-difluoromethylornithine

In the testosterone-induced hypertrophic and antifolate (N¹⁰-propargyl,5,6-dideazafolic acid, CB 3717)-induced hyperplastic mouse kidney models, a marked increase of two diamine levels — putrescine and cadaverine — occurred which paralled induced ornithine decarboxylase (ODC) activity. Under these conditions the augmentation of spermidine levels was much smaller, while spermine levels were affected differentially — increased by testosterone and decreased by CB 3717; this resulted in an increase of spermidine/spermine ratio in hyperplastic, but not hypertrophic kidney. α-Difluoromethylornithine (DFMO) prevented testosterone- or CB 3717-induced increment of both diamine levels. Spermidine and spermine depletion in response to DFMO was significant in hyperplastic kidney only. DFMO also significantly affected the other biochemical markers of hyperplasia, namely lowered CB 3717-induced cell proliferation rate and increased S-adenosylmethionie decarboxylase (AdoMetDC) activity. In contrast, testosterone-induced hypertrophy was not influenced by DFMO, as judged by the lack of its effect on S-adenosylmethionine synthetase and cystathionine synthase activity. These results indicate that the increase of putrescine levels does not mediate testosterone-induced renal hypertrophy and possibly also antifolate-induced hyperplasia. The involvement of spermidine in mediation of renal hyperplasia is highly possible, while that of spermine is excluded.     

Effect of 1-amino-oxy-3-aminopropane on polyamine metabolism and growth of L1210 cells.

1-Amino-oxy-3-aminopropane (AOAP) was reported to inhibit several mammalian polyamine-biosynthetic enzymes in vitro, including ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) [Khomutov, Hyvönen, Karvonen, Kauppinen, Paalanen, Paulin, Eloranta, Pajula, Andersson & Pösö (1985) Biochem. Biophys. Res. Commun. 130, 596-602]. In order to clarify its mechanism of action in intact cells, the inhibitory properties of AOAP on the growth and polyamine metabolism of L1210 cells were compared with those seen in a variant subline (D-R cells) which overproduces ODC. As little as 20 microM-AOAP completely blocked proliferation of L1210 cells, and this effect was reversed by the concomitant addition of exogenous putrescine or spermidine. Growth of D-R cells was not affected by AOAP at concentrations up to 0.5 mM. There was no difference in the uptake of AOAP between the L1210 and the D-R cells. Exposure of L1210 or D-R cells to AOAP greatly decreased ODC activity in undialysed cell extracts, but did not decrease AdoMetDC. Activities of both enzymes were increased severalfold by AOAP treatment when activity was measured in dialysed extracts. Treatment with AOAP depleted intracellular putrescine and spermidine contents of L1210 cells, while inducing a massive accumulation of decarboxylated AdoMet. The 8-fold higher putrescine pool present in untreated D-R cells was depleted in a dose-dependent manner by AOAP, but a significant decrease in spermidine and accumulation of decarboxylated AdoMet required 10 times higher drug concentrations, and the changes were much less dramatic than in L1210 cells. These results indicate that in L1210 cells AOAP behaves primarily as a reversible inhibitor of ODC.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5' leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiations. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.     

Growth and polyamine metabolism inPyrenophora avenae exposed to cyclohexylamine and norspermidine

Summary The effectiveness of inhibitors of polyamine biosynthesis in controlling plant pathogenic fungi is well established. The spermidine synthase inhibitor cyclohexylamine (CHA) and the spermidine analogue norspermidine were evaluated againstin vitro growth of the oat stripe pathogenPyrenophora avenae. Mycelial growth was reduced by 55% upon exposure to 2.0mM CHA while the same concentration of norspermidine reduced growth by 63%. Neither inhibitor had any effect on ODC or AdoMetDC activities, nor the flux of label from ornithine through to the polyamines. Levels of free polyamines in fungal tissue exposed to 0.01 mM norspermidine were unaltered, although 1.0mM CHA did produce a 75% increase in fungal putrescine content. These data suggest that CHA and norspermidine do not reduce fungal growth as a result of a perturbation in polyamine biosynthesis.Abbreviations ODC ornithine decarboxylase - ADC arginine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO adifluoromethylornithine - CHA cyclohexylamine     

Methyl jasmonate alters polyamine metabolism and induces systemic protection against powdery mildew infection in barley seedlings

Treatment of the first leaves of barley (Hordeum vulgare L. cv. Golden Promise) seedlings with methyl jasmonate (MJ) led to small, but significant increases in levels of free putrescine and spermine 1 d later and to significant increases in levels of free putrescine, spermidine and spermine by 4 d following treatment. MJ-treated first leaves also exhibited significant increases in the amounts of soluble conjugates of putrescine and spermidine 1, 2 and 4 d after treatment. In second leaves of plants where the first leaves had been treated with MJ, no significant changes in levels of free polyamines were observed, but significant increases in levels of soluble conjugates of putrescine and spermidine were detected. These changes were accompanied by increased activities of soluble ornithine decarboxylase (ODC), soluble and particulate arginine decarboxylase (ADC), and S-adenosylmethionine decarboxylase (AdoMetDC), in first and second leaves following treatment of the first leaves with MJ. Activities of soluble and particulate diamine oxidase (DAO) were also higher in first and second leaves following treatment of the first leaves with MJ. Treatment of the first leaves with MJ led to a significant reduction in powdery mildew (Blumeria graminis f. sp. hordei) infection on the second leaves and also resulted in significant increases in activities of the plant defence-related enzymes, phenylalanine ammonia lyase (PAL) and peroxidase.     

The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step.

Four mouse and two human tumour cell lines resistant to alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), were analysed for the activities of polyamine-biosynthetic and -biodegradative enzymes as well as for cellular polyamine contents. In all but one of these cell lines the resistance to DFMO was based on an overproduction of ODC. In a human myeloma cell line the resistance was based on a greatly enhanced arginase activity. Except for one L1210 variant cell line, all the resistant cell lines contained elevated S-adenosylmethionine decarboxylase activity. Similarly, all the resistant mouse, but not human, cell lines displayed enhanced spermidine and spermine synthase activities. Arginase activity was detected only in human cell lines. In both DFMO-resistant cell lines the activity of arginase was strikingly elevated. Of the biodegradative enzymes, polyamine oxidase activity was readily detectable in all mouse cells, but no measurable activity was found in the human cells. Spermidine/spermine N1-acetyltransferase activity was elevated in three out of four resistant mouse cell lines. Even though the concentration of spermidine was usually lower in the overproducer cells, this was compensated by an increased content of spermine. The two resistant human myeloma cells contained intracellular ornithine concentrations that were from more than 5 to more than 20 times higher than those in the parental cells.     

Early events in the response of fast skeletal muscle to chronic low-frequency stimulation. Polyamine biosynthesis and protein phosphorylation.

The concentrations of putrescine, spermidine and spermine and the activities of ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAM-D) were investigated in fast muscle subjected to chronic low-frequency electrical stimulation. Both ODC and SAM-D activities increased markedly between 18 and 48 h of stimulation. Changes in enzyme activities were followed by phasic elevations in the concentrations of putrescine, spermidine and spermine. Peak levels were reached first by putrescine at 3-4 days, followed by spermidine at about 9 days and then by spermine at about 11 days. A possible relationship was sought between these events and changes produced in vitro in the phosphorylation pattern of cytoplasmic proteins and the total activity of cyclic AMP-dependent protein kinase. However, during the early stages of stimulation, no prominent changes were seen either in the phosphorylation pattern or in the activity of cyclic AMP-dependent protein kinase. These characteristics changed significantly at a later stage (by 12 days of stimulation) and became indistinguishable from those of slow muscle by 3 to 4 weeks of stimulation.     

Ornithine decarboxylase, transglutaminase, diamine oxidase and total diamines and polyamines in maternal liver and kidney throughout rat pregnancy.

Ornithine decarboxylase (ODC; EC 4.1.1.17), transglutaminase (EC 2.3.2.13), diamine oxidase (DAO; EC 1.4.3.6) and total di- and poly-amines were studied in rat liver and kidney cortex throughout pregnancy. In liver, ODC activity exhibited two major peaks (4.5-5 times the control activities) on days 15 and 17. Also putrescine and spermidine increased biphasically (3-4-fold), but no variation in spermine content was observed. Transglutaminase activity showed slight variations only near the end of gestation. In kidney, ODC activity did not fluctuate significantly during pregnancy, whereas both transglutaminase activity and putrescine content showed three major increases, in very early, middle and late pregnancy. No significant variations in spermidine and spermine were observed. In both organs, DAO activity, very low or undetectable until day 10, dramatically increased (10- and 20-fold in kidney and liver respectively) in the second half of pregnancy, reaching maxima on days 16-17 and 19. The results obtained for transglutaminase, ODC and total di- and poly-amines are interpreted on the basis of hyperplastic and hypertrophic events in the liver and kidney respectively. The behaviour of DAO suggests that the enzyme plays an important role in the control of intracellular diamine concentration.     

Reversal of the growth inhibitory effect of α-difluoromethylornithine by putrescine but not by other divalent cations

Summary Treatment with -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), depletes the putrescine and spermidine content, and reduces the growth rate of Ehrlich ascites tumor cells.The addition of putrescine, which is the immediate precursor of spermidine, promptly replenished the intracellular putrescine and spermidine pools and completely reversed the antiproliferative effect of DFMO. A sequential accumulation of spermine, spermidine and putrescine was observed.1,3-diaminopropane, a lower homolog of putrescine, did not reverse the antiproliferative effect of DFMO, despite its structural similarity and identical positive charge. By inhibiting remaining ODC activity, resistant to 5 mM DFMO, and possibly by inhibiting spermine synthase activity, 1,3-diaminopropane produced a further decrease in total polyamine content by reducing the spermine content.Mg²⁺, which can replace putrescine in many in vitro reactions, completely lacked the capacity to reverse the antiproliferative effect of putrescine and spermidine deficiency.Abbreviations DFMO -difluoromethylornithine - ODC ornithine decarbxylase     

Effects of diethyldithiocarbamate and endogenous polyamine content on cellular responses to hydrogen peroxide cytotoxicity.

In exponential-phase Chinese-hamster cells, 0.1 mM-diethyldithiocarbamate (DDC) afforded greater than 1 log survival protection to cultures treated before and during exposure to 1 mM-H2O2. Both DDC and H2O2 treatment stimulated the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, within 4 h of exposure. DDC, and to a lesser degree H2O2, also stimulated the activity of spermidine N1-acetyltransferase (SAT), the rate-limiting enzyme in polyamine catabolism. The increase in SAT activity, after exposure to DDC or another stress (heat shock), was inhibited in cells depleted of putrescine and spermidine by alpha-difluoromethylornithine (DFMO), the enzyme-activated suicide inhibitor of ODC. Pretreatment with DFMO or heat shock also induced resistance to H2O2 cytotoxicity. Since SAT activity is low in resting cells, yet stimulation of enzyme activity depends on endogenous spermidine pools, these results suggest that the expression of SAT activity occurs by a mechanism involving a stress-dependent displacement of spermidine into a new intracellular compartment. The stimulation of ODC and SAT activities does not appear to be a necessary component of the mechanism by which DDC protects cells from H2O2 cytotoxicity, although spermidine displacement may be a common facet of the cellular response to stress.