Accumulation of Decarboxylated S-Adenosyl-L-Methionine in Mammalian Cells as a Consequence of the Inhibition of Putrescine Biosynthesis

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1 Eur. J. Biochem. 123, 4954 (1952) FEBS 1982 Accumulation of Decarboxylated SAdenosylLMethionine in Mammalian Cells as a Consequence of the Inhibition of Putrescine Biosynthesis Pierre S. MAMONT, Charles DANZIN, Joseph WAGNER, Mirlyse SIAT, AnneMarie JODEROHLENBUSCH, and Nicole CLAVERI E Centre de Recherche Merrell International, Strasbourg (Received November 24, 1981) Biological transmethylation reactions and polyamine biosynthesis share the substrate SadenosylLmethionine. Under normal conditions, decarboxylated SadenosylLmethionine, the aminopropyl donor for polyamine biosynthesis, does not accumulate because of its rapid utilization in spermidine and spermine synthesis. Alteration of polyamine synthesis by DLadifluoromethylornithine, an enzymeactivated irreversible inhibitor of Lornithine decarboxylase, leads to a striking accumulation of decarboxylated SadenosylLmethionine in rat hepatoma cclls cultured in vitro and in rat ventral prostate. This increase is due both to lack of putrescine and spermidine for the aminopropyltransferase reactions and to the elevation of SadenosylLmethionine decarboxylase activity. The biological implications of accumulation of decarboxylated SadenosylLmethionine are discussed with regard to the regulation of SadenosylLmethionine decarboxylase activity and to the antiproliferative effects of DLadifluoromethylornithine. Biological transmethylation reactions and polyamine biosynthesis share the substrate SadenosylLmethionine (AdoMet). Decarboxylation of AdoMet catalyzed by SadenosylLmethionine decarboxylase leads to Sadenosyl(5 ) deoxy(5 )3metl~ylthiopropylamine (decarboxylated Ado Met) [I 31 which does not accumulate in cells and organs due to its rapid consumption in reactions catalyzed by the aminopropyltransferases (spermidine and spermine synthases) [4]. We now report that inhibition of polyamine biosynthesis by madifluoromethylornithine (FzMeOrn), an enzymeactivated irreversible inhibitor of Lornithine decarboxylase [S], dramatically increases decarboxylated AdoMet levels in rat hepatoma tissue culture (HTC) cells and in rat ventral prostate. The accumulation of decarboxylated AdoMet parallels the increase of SadenosylLmethionine decarboxylase activity which, as previously demonstrated, is a consequence of depletion of intracellular polyamines [6] and particularly of spermidine depletion [7 11. The biological implications of the accumulation are discussed. MATERIALS AND METHODS mxdifluoromethylornithine (F2MeOrn) [S] was dissolved in phosphatebuffered saline and the solution was adjusted to ph 7.4 with 1 M NaOH. Putrescine dihydrochloride, spermidine trihydrochloride, SadenosylLmethionine and Sadenosylhomocysteine were purchased from Sigma Chemical Co. (St LOUIS, MO, USA). 1,3,6Triaminohexane and 1,4,7triaminoheptane were synthetized in our Centre by P. Bey. Decarboxylated SadenosylLmethionine was synthesized in our Centre by M. Kolb following the procedures described in the literature [I 1,121; DL[114C] Abhre1Jiation.s. HTC cells, hepatoma tissue culture cells; AdoMet, SadenosylLmethionine; decarboxylated AdoMet, Sadenosyl5 deoxy (5 )3niethylthiopropylamine; FzMeOrn, madifluoromethylornithine. Enzymes. LOrnithine decarboxylase (EC ) ; Sadenosyl1. methionine decarboxylase (EC ). Ornithine (58 Ci/mol) and SadenosylL[l 4C]methionine (6 Ci/mol) were purchased from Radiochemical Centre (Amersham, Bucks, UK). Rut Hepatornu Tissue Culture (HTC) Cells Rat hepatoma tissue culture (HTC) cells were routinely grown as previously described [7]. Dialysed horse serum (Gibco) (1 /, viv) replaced newborn calf serum in experiments with ornithine decarboxylase inhibitors and polyamines. Cell growth and cell viability were measured by cell counting in a haemocytometer in the presence of Trypan blue. For AdoMet decarboxylase assay, cells (lo ) were harvested by centrifugation, washed with cold phosphatebuffered saline and sonicated in.7 ml of buffer consisting of 1 mm sodium phosphate buffer, ph 7.2, 2.5 mm dithiothreitol and.1 mm EDTA. Activity was determined on crude sonicated cell extracts by the procedure developed by Pegg and WilliamsAshman [I] as previously described [7]. Proteins were determined by a modification [13] of the method of Lowry [I41 with bovine serum albumin as standard. For the determination of polyamines and of AdoMet and its metabolites, 2 x 1 cells (5.5 mg protein) were disrupted by sonication in 1.8 ml.5 M HzS4 and after precipitation of proteins by.2 M HC14, aiialyses wcrc performed directly on 1 1pl aliquots of the supernatants. Animuls Male rats of the SpragueDawley strain ( g body weight, purchased from Charles River, France) had frce access to standard diet and were given water (id lihirim under a constant 12h light, 12h dark lighting schedule. DLaDifluoromethylornithine dissolved in water was given by gavage (2 mg/kg body weight). Rats given water by gavage served as controls. Animals were killed by decapitation at the same time of day to minimize effects due to diurnal fluctuations. For decarboxylase assays, the ventral prostates were excised immediately after sacrifice, weighed and homogenized

2 5 with 5 vol. 3 mm sodium phosphate buffer ph 7.1 containing.1 mm EDTA,.25 M sucrose,.1 mm pyridoxal phosphate and 5 mm dithiothreitol. Ornithine and AdoMet decarboxylase activities were measured according to Ono et al. [I51 and Janne and WilliamsAshman [I61 respectively. For determination of AdoMet and its metabolites, the ventral prostates were freed of fat and connective tissue, weighed and frozen in liquid nitrogen. Then they were homogenized at 4 C with 5 m1.2 M HC14. After centrifugation (3 x g), analyses were performed in the supernatants. Determination of AdoMet and Its Metabolites These were measured by the reversephase ionpair highperformance liquid chromatography procedure of Wagner et al. [17]. The chronlatographic system consisted of two model 6 A pumps, a M66 solvent programmer, a M44 ultraviolet absorbance detector operating at 254 nm and an automatic sample injector WISP 71A, all from Waters Assoc. (Milford, MA, USA). The column was a pbondapak column (1 pm particle size, 3.9 x 3 mm). Signals were recorded with a SP 41 digital integrator from Spectra Physics (Santa Clara, CA, USA). The elution system was prepared from two buffers: buffer A,.1 M NaH2P4 (ph 2.5)/acetonitrile (94: 6, v/v) containing 8 mm sodium octane sulfonate (Eastman Kodak Co., Rochester, NY, USA); buffer B,.1 M NaHZP4 (ph 3.l)/acetonitrile (7: 3, v/v), 8 mm sodium octane sulfonate. A linear gradient was preparated from 85% buffer A and 15% buffer B to 15% buffer A and 85 o/, buffer B within 3 min. Buffer flow rate was 1.5 ml/min and the temperature of the column was 4 "C. The lower limit of detection of AdoMet and its metabolites was of the order of 5 pmol. Determination of Polyumines Polyamines were determined by highperformance liquid chromatography either simultaneously with AdoMet determination by fluorescence detection after postcolumn derivatization with ophthaldehyde [17] or using the elution system of Seiler and Knodgen [IS], except that a linear gradient was used. RESULTS Effects of adifluoromethylornithine on HTC Cell SAdenosyl~methionine Decarhoxylase Activity, Polyamine and Decarhoxyluted SAdenosyl1.methionine Contents We have previously reported that FzMeOrn increases Ado Met decarboxylase activity in HTC cells after a lag period of 9 12 h.this lag period has been attributed to the time needed to achieve sufficient spermidine deficiency [lo]. As illustrated in Fig. 1, in parallel with this elevation of decarboxylase activity, decarboxylated AdoMet which was barely detectable in control cells (.1 nmol/mg protein), accumulated to reach a concentration about twice as high as that of AdoMet. FzMeOrn did not affect the increase of AdoMet content which followed induction of cell proliferation, whereas a 4 reduction of the Sadenosylhomocysteine content was obobserved [control cells: 57 k 5 (S.E.M.) pmol/mg protein; FzMeOrntreated cells: 34 k 3 (S.E.M.) pmol/mg protein]. Another peak appeared in the chromatographic profiles of perchloric acid extract of cells incubated in the presence of FzMeOrn. The chemical nature of this ultravioletabsorbing material has not yet been characterized [17] Time (days) Fig. 1. Effect oj FZMeOrn on HTC cell AdoMet decarhoxyluse and intracellulur AdoMet nnd decarboxylated AdoMet contents. HTC cells were incubated from zero time in the absence (, A, ) or in the presence (, A, m) of 5 mm FzMeOrn. AdoMet decarboxylase activities (, ). AdoMet contents (A, A) and decarboxylated AdoMet contents (, m) were determined in duplicate at the indicated time; duplicate samples differed by no more than 1 "/, Reversal of the Effects of adifluoromethylornithine by Polyamines and Triamines If the increase of decarboxylated AdoMet levels resulted from inhibition by FzMeOrn of putrescine synthesis and the increase of AdoMet decarboxylase activity, we should be able to reverse these effects by restoration of the normal content of intracellular polyamines. As illustrated in Fig. 2, addition of 1 pm spermidine to the culture medium of HTC cells incubated in the presence of 5 mm FzMeOrn for 24 h replenished the intracellular levels of spermidine and decreased AdoMet decarboxylase activity, confirming previous observations [8 11. Concomitantly decarboxylated AdoMet content returned towards control values (Fig. 2). Two synthetic triamines 1,3,6triaminohexane and 1,4,7 triaminoheptane, which reversed the antiproliferative effects of FZMeOrn (Table I), analogous to the effect of spermidine, were used in comparison with spermidine. 1,4,7Triaminoheptane (1 pm) decreased AdoMet decarboxylase activity and reduced decarboxylated AdoMet accumulation after a lag period of about 2 h (Fig. 2, see also Table 2). In contrast, at all intracellular concentrations used, 1,3,6triaminohexane decreased neither AdoMet decarboxylase activity to control values nor the decarboxylated AdoMet content of the cells (Table 2), even 24 h after its addition (results not shown). When used in 1 mm concentration, the intracellular content of 1,3,6triaminohexane was equivalent to that resulting from the addition of 1 pm 2,4,7triaminoheptane to the culture medium (Table 2). The addition of 1 pm putrescine to the medium of cells treated with FzMeOrn for 48 h resulted in a transient abovenormal cellular putrescine content and in a further increase of AdoMet decarboxylase activity (Fig. 3). Thereafter, and as previously reported [lo], decline of the putrescine content and of AdoMet decarboxylase activity paralleled the restoration of the spermidine content of the cells, whereas spermine content returned to control values at a later time (8 h). An hour after addition of putrescine, the decarboxylated AdoMet content of the cells was decreased from 1.5 to.25 nmol/mg

3 ~ [L H c OI v Q Qi 2 4 6,... c ) c L Q m 1 E. E ).. 5 E 5 n ). Putrescine l5 [E. Spermidine l5 r F. Spermine 15 I. Triaminoheptane Time (h) Fig. 2. Effect of spermidine and triamine addition on (A) AdoMet decarhoxylase activity and intracellular contents of (C) AdoMet, (B) decarhoxylufed AdoMet and (DG) polyumines of F2MeOrntrealed cells. HTC cell cultures (1.1 x los cells/nil) were preincubated in the presence (, A. A) oiabsence () of 5 nim F2MeOrn for 24 h. At that time, 1 pm spermidine (A) or I pm 1,4,7triaminoheptaiie (A) were added to portions of FzMeOrntreated cells. (A) AdoMet decarboxylase activities and (C) AdoMet, (B) decarboxylated AdoMet and (DG) polyamine contents were determined as described in Materials and Methods. Values given for intracellular triaminoheptdne were estimates because of the poor chromatographic rcsolution from residual spermidine content (.5 nmol/mg protein) Table 1. Reversalqf file antiprol~erativc~effectsoff~mcorn bypolyamines and triamines HTC cell cultures were incubated in the presence of absence of F2MeOrii (5 nm) for 24 h. At that time, 1 pm putrescine, spermidine, 1,3,6triaminohexane or 1,4,7triaminoheptane were added to portions of Fe2MeOrntreated cell culture. Cell numbers were measured as cells/ml ciiltiir~... Additions x cell number, at Oh 24 h 48 h 12 h ml' None (control) FzMeOrn FzMeOrn putrewne FnMeOrn + spermidine F2McOrn + triaminohcptane FzMeOrn + ti ldminohexane The effect on AdoMet levels was not observed by addition of sperrnidine or of triamines (Fig. 2 and Table 2). Efrects of adif7uoromethylornithine on the Activities Of Prostatic Ornithi'' Decarboxyluse and SAdenosylLmethionine Decarhoxylase and on Decarbox~lutedSAd~no,~ylLmethionine Content As illustrated in Fig. 4, following a single dose (2 mg/kg; p..) of FzMeOrn, prostatic ornithine decarboxylase activity decreased to about 14% of control values within 6 h, confirming a previous report [19]. Thereafter, the activity slowly returned to 5 % of control values 24 h after the administration of F2MeOrn. An inverse pattern was observed for AdoMet decarboxylase activity which was elevated threefold over control values within 12 h and then was slightly decreased. In parallel with the increase of AdoMet decarboxylase activity a ninefold increase of the decarboxylated AdoMcl content of the organ was noticed, while the AdoMet content (77 8 nmol/g wet weight) remained fairly constant (not shown). protein. Thereafter, a slow return to basal control values was observed. AdoMet concentration was also decreased but this decrease was followed by an overshoot which reached levels about three times those in control and F2MeOrntreated cells. DISCUSSION Under normal conditions, the amount of decarboxylated AdoMet in rat ventral prostate is smaller by at least one or two orders of magnitude than that of AdoMet. These findings are in complete agreement with those of Hibasami

4 ~ ~~~~ ~ ~~ ~~ ~. ~ ~~ ~ 52 Table 2. Effects of addition of spermidinr or triaminrs on AdoMei decarbox$ase activity, AdoMet, drcarhoxylated AdoMet and polyumine contents of' FzMeOrntreated HTC cells HTC cell cultures (lo5 cells/ml) were incubated for 24 h in the presence of absence of 5 mm FzMeOrn. At that time F2MeOrntreated cell cultures received I pm spermidine, 1 pm 1,4,74riaminoheptane, 1 pm or 1 mm 1,3,6triaminohexane and the cultures were further incubated for 6 h. Polyamines, AdoMet and decarboxylated AdoMet contents were measured on perchloric acid extracts and AdoMet decaiboxylase activities on cell extracts prepared as described in Materials and Methods. Results are the average of two separate experiments Additions AdoMet AdoMet Decarb Putrescme Spermidine Spermine Triamines decarboxylase oxylated AdoMet nmol COz h' mg protein' FZMeOrn (at zero time) 8. FzMeOrn (at 6 h) 12.3 FzMeOrn + spermidine 1.1 F2MeOrn + triaminoheptane 4.2 FzMeOrn + triaminohexane" 11.4 FZMeOrn + triaminohexaneb 11.6 None (control) 2.1 nmol/mg protein < <o * 1 pm. 1 mm. 3 C \ \ \ e, I [ D. Putrescine 2, F. Spermine 15 m E Time (h) Fig. 3. Eff;.ct ofputrrscine addition on (A) AdoMet decarhoxylase and intracrllular contents of (C) AdoMet, (BJ drcctrhoxyluted AdoMet and (D F) pofyamines oj F&eOvntreutc,d cells. HTC cell cultures were preincubated in the presence (, A) or absence () of 5 mm FezMeOrn for 48 h. 1 pm putrescine (A) was added to portion5 of FZMeOrntreated cells and (A) AdoMet decarboxylase activities, (C) AdoMet, (B) decarboxylated AdoMet and (D F) intracellular polyamine concentrations were determined et al. [4] (see also [2]). In HTC cells the concentration of decarboxylated AdoMet is estimated to be less than 2% of the AdoMet content. We demonstrate now that depletion of putrescine and spermidine causes, in parallel with the elevation of AdoMet decarboxylase activity, a marked increase of the decarboxylated AdoMet content in HTC cells and in rat prostate. Similar observations were recently reported by Pegg et al. [21] for SV43T3 fibroblasts treated with FzMeOrn. That accumulation of decarboxylated AdoMet in HTC cells probably results from substrate limitation for the spermidine and spermine synthase reactions, as well as from the increase of AdoMet decarboxylase activity, is demonstrated by restoring the intracellular contents of putrescine and spermidine. The decrease of the decarboxylated AdoMet content after addition of putrescine to the culture medium is readily explained by

5 6678. I l l I I Time after administration (h) t E l Fig.4. $~ts o/ FzMeOrn on rut prosirufc ornifhine and AdoMet decarhox~kuse.~ arid derurboq'lated Adohlet wntenf. FzMeOrn (2 mg/kg body weight) was administered by gavage at zero time. At the indicated times, animals were killed and enzyme activities and decarboxylated AdoMet (A) contents werc determined as described in Materials and Methods. Enzyme activities were expressed as percentages of control values: () ornithine decarboxylase, 31 i 6 nmol C2 h' g'; () AdoMet decarboxylase nmol h' g'; values are means +_ S.E.M. of five animals the facts that putrescine is cosubstrate with decarboxylated AdoMet of the spermidine synthase reaction and that the conversion of putrescine to spermidine is accompanied by a decline of AdoMet decarboxylase activity. Addition of sperinidine also decreases this activity. As a consequence, but also because of its consumption by the spermine synthase reaction, decarboxylated AdoMet content returns to control values. Addition of the triamine 1,4,7triaminoheptane decreases AdoMet decarboxylase activity and consequently stops the accumulation of decarboxylated AdoMet. In contrast to the addition of spermidine, decarboxylated AdoMet levels did not return to basal control values. These findings suggest that this triamine is not a substrate of spermidine and spermine synthases and reinforce the idea that accumulation of decarboxylated AdoMet content caused by FzMeOrn is due both to the lack of the substrates for aminopropyltransferase enzymes and to the increase of AdoMet decarboxylase activity. Comparison of the effects of 1,4,7triaminoheptane with spermidine rules out a possible and essential direct or indirect participation of decarboxylated AdoMet in the elevation of AdoMet decarboxylase activity measured in dialyzed extracts of F2MeOrntreated cells. Indirect evidence for stabilization of AdoMet decarboxylase after FzMeOrn treatment and dcstabilization after restoration of the normal spermidine content of the cells has been presented [9,1]. The fact that 1,4,7triaminoheptane decreases AdoMet decarboxylase activity without depleting the cells of decarboxylated AdoMet argues strongly in favor of a polyamine effect. This, together with the ineffectiveness of 1,3,6triaminohexane to affect AdoMet decarboxylase activity, further supports the notion of a specific regulatory role of spermidine in the control of this enzyme [9,1]. The finding that decarboxylated AdoMet accumulation to such an extent may be indicative of slow catabolism and(or) slow excretion. At present, nothing is known about these parameters, but inhibitors of ornithine decarboxylase may prove very valuable in elucidating these problems. In spite of the marked increase of the decarboxylated AdoMet content elicited by FzMeOrn, AdoMet levels remain constant in HTC cells. During our study, the only condition which leads to a decrease in AdoMet levels is the addition of putrescine to the culture medium of F2MeOrntreated cells. This is probably due to activation of AdoMet decarboxylase in situ by the abovenormal content of putrescine which permits sufficient synthesis of decarboxylated AdoMet for immediate conversion of putrescine to spermidine. Thus 2.7times more spermidine has been formed than decarboxylated AdoMet consumed within 2 h. However, decrease of AdoMet levels not only ceases once the spermidine levcl has been restored, but AdoMet overaccumulates relative to control values at a later time. Taken together, these findings may reflect induction by polyamine starvation of compensatory mechanism at the level of AdoMet synthesis or degradation and/or may suggest a decreased utilisatioii due to reduction of methylation reactions. These aspects certainly deserve future consideration. Possible roles of decarboxylated AdoMet as a rcgulator of AdoMet metabolism are still an open question. Some studies in vitro have indicated that decarboxylated AdoMet can inhibit AdoMet decarboxylase [ and, whcn present in high concentration, the spermidine synthase reaction [25,26]. Furthermore, decarboxylated AdoMet has been noticed to inhibit in vitro rat liver AdoMet lyase [27] and brain methylene tetrahydrofolate reductase [28]. Little information is presently available concerning possible interferences of decarboxylated AdoMet in transmethylation reactions. Decarboxylated AdoMet does not appear to exert profound inhibitory activity in vitro towards protein carboxylmethyltransferase [29], catechol methyltransferase, phenylethanolamine Nmethyltransferase, hydroxyindole Omethyltransferase [3] or acetylserotonin methyltransferase [313. Some inhibition of histamine Nmethyltransferase in vitro has, however, been reported [3,31]. Furthermore, its role as a methyl donor has not yet been extensively investigated [29, 311 and its effect on nucleic acid methylations is completely unknown. The fact that 1,3,6triaminohexane can reverse the antiproliferative effects of F2MeOrn in HTC cells without affecting the elevated content of decarboxylated, AdoMet again strongly suggests that reduction of cell growth rates by F2MeOrn essentially results from limitation of polyamine biosynthesis and not from accumulation of decarboxylated AdoMet. However, it cannot be considered establishcd that in other circumstances, as for instance in the ventral prostate [32] where decarboxylated AdoMet is increased up to 25fold over AdoMet content after 3days oral administration of FzMeOrn, part of the effects off2meorn may hot be attributed to either direct effects of decarboxylated AdoMet on AdoMet inetabolism or to a blockade of methylthioadenosinc synthesis. 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