CELL BIOCHEMISTRY AND FUNCTION VOL.
10: 243-250 (1992)
Ornithine Decarboxylase and Ornithine Decarboxylaseinhibiting Activity in Rat Thymocytes CLAUDIO STEFANELLI, CARMEN ROSSONI, FABRIZIA FERRARI, FLAVIO FLAMIGNI AND CLAUDIO M. CALDARERA Department of Biochemistry ‘G. Moruzzi’, University of Bologna, 40126 Bologna, Italy
Isolation of thymocytes from rat thymus resulted in the disappearance of the high activity of ornithine decarboxylase (ODC) that characterizes the thymus of young rats, together with the appearance of an antizyme-like ODC inhibiting activity, which showed a chromatographic profile that resembled that of dexamethasone-treated rat thymus. Omission of serum or addition of dexamethasone or spermidine did not affect appreciably the extent of the antizymelike activity. On the other hand, a variety of hormonal effectors, i.e. insulin, glucagon, adrenalin and T3, as well as the phorbol ester, PMA or the mitogen, concanavalin A (Con A) induced ODC activity in cultured thymocytes together with the disappearance of the antizyme-like activity. A paradoxical, transient induction of ODC was caused by the transcriptional inhibitor, actinomycin D. Complexed ODC was detected in rat thymus, but not in thymocytes, either quiescent or stimulated by mitogens. These results indicate that thymic lymphocytes can express either ODC activity or its inhibitor depending on the hormonal and proliferative status of the cells. KEY
WORDS--0rnithine decarboxylase; antizyme; thymocytes; polyamines.
ABBREVIATIONS-ODC, ornithine decarboxylase; Con A, concanavalin A; HPLC, high performance liquid chromatography; PMA, phorbol- 12-myristate-13-acetate; T3, 3,5,3’-triiodothyronine.
INTRODUCTION The importance of the polyamines, putrescine, The relevance of each particular regulatory spermidine and spermine, in cell growth and differ- mechanism of ODC appears to depend on the entiation is reflected by the complexity of the specific stimuli and the cell type. In lymphoid cells, regulation of ornithine decarboxylase (ODC), the marked increases in ODC levels are observed rate limiting enzyme in polyamine biosynthesis. following activation with mitogens, antigens or Transcriptional, translational and post-transla- phorbol esters.”-16 Active ODC and polyamines tional control of ODC has been described,’,’ as are required for the interleukin-2-dependent well as its interaction with a specific protein inhibi- growth and alloantigen activation of cytolytic tor, named a n t i ~ y m eAlthough .~ antizyme was ini- T-lymphocyte~’~~” and are involved in the down tially discovered in tissues or cells exposed to high regulation of the interleukin-2 production by T concentrations of polyamines: antizyme mRNA cells in EL-4 lymphoma cells.I8 and ODC-antizyme complex were subsequently Changes in ODC activity also occur in lymphoid found to be present constitutively in several rodent organs, such as the thymus and spleen, in relation Furthermore, it has been proposed that to their trophic and functional state. Thus, ODC ODC complexed to antizyme is not only catalyti- activity is very high in the thymus of young anically inactive, but also rapidly degraded.” mals, but declines with age, as the organ undergoes i n v o l ~ t i o n . ’ODC ~ activity is increased in lymphoid organs stimulated not only by mitogens but also by hormones, such as insulin, catecholamines, Conversely, Address for correspondence: Dr Claudio Stehnelli, Diparti- growth hormone and pr~lactin.’~-’~ mento di Biochimica, via Irnerio 48, 40126 Bologna, Italy. ODC levels decrease dramatically in the thymus 0263-6484/92/340243-08%09.00 01992 by John Wiley & Sons, Ltd.
244
C. STEFANELLI ET AL.
and spleen of animals injected with dexamethasone, preceding by several hours the cytolytic effect of the glucocorticoid.26 In particular, in the thymus of rats treated with dexamethasone, the reduction in the content of ODC mRNA could account only partially for the marked decrease in ODC activity.” However, dexamethasone caused the appearance of an antizyme-like inhibitor of ODC in thymus and spleen extract^.^^*^^ In the present study, we have investigated conditions and stimuli leading to the expression of ODC and antizyme activity in isolated thymic lymphocytes (thymocytes).
One unit of ODC activity is defined as the amount releasing 1 nmol CO, h-’. For the determination of ODC inhibitory activity6 cell extracts were tested for their ability to inhibit a fixed amount of ODC from rat thymus that had been partially purified by ammonium sulphate precipitation followed by high performance ion exchange chromatography on a Bio-Gel TSK-DEAE-5-PW column and gel filtration on a Bio Sil TSK-250 column. One unit corresponds to the amount inhibiting 1 unit of ODC activity. The amount of complexed ODC was measured by assaying in 40 pl of extract the enzyme activity releasable by an excess of rat thymus ODC inactivated by a-difluoromethyl ornithine (DFMO) as MATERIALS AND METHODS previously described.6 High performance gel filtration chromatography Chemicals of ODC-inhibiting activity was performed on a The chemicals for preparation of culture media TSK-250 600 x 7.5 mm column calibrated with were obtained from Gibco Laboratories. Biochemi- standard proteins of known Mr. For this determicals were from Sigma Chemical Co. and other nation about lo8 cells were lysed in 0-8ml of lysis buffer. After centrifugation, 0.5 ml of crude extract laboratory chemicals were products of E. Merck. was injected and eluted with 50 mM Tris-HC1 pH 7.0 containing 0.3 M NaCl, 0.1 mM EDTA and Animals and Cell Cultures 1 mM dithiothreitol at a flow rate of 0.5 ml minFractions of 0.5 ml were collected and 4Opl of Male Wistar rats were used. Thymocytes were every fraction were tested for ODC-inhibitory acobtained from the thymuses of 4 to 10 week-old animals. Thymus glands were removed, washed in tivity, using a fixed amount of purified ODC giving phosphate-buffered saline and immersed in a stan- 2500 dpm (assay)- in the absence of inhibitors. dard medium containing RPMl 1640 with 10 per Rat thymus extracts were prepared as previously cent fetal bovine serum, 2 mM glutamine, 25 mM de~cribed.’~ HEPES, penicillin and streptomycin (100 units ml-l). Thymuses were chopped into small Other Assays pieces and disaggregated. The cell suspension was For the determination of polyamines the pelleted filtered and centrifuged at 800 g for 5 min. The cells were resuspended in 0.5 ml of chilled 0.3 M pelleted cells were resuspended in 2 ml of standard PCA and homogenized by repeated freezing and medium, counted and diluted to lo7 cellsml-’. thawing. After centrifugation polyamines were Cell suspensions (5 ml) were incubated in sterile flasks in a humidified atmosphere of 5 per cent CO, measured in acid extracts by HPLC.30 Spermidine acetyltransferase activity was measured in the same in air at 37°C. extracts used for the ODC assay by measuring the incorporation of labelled acetyl coenzyme A into ODC and ODC-Inhibitory Activities per mi dine.'^
’.
At the time indicated after treatment, the cells were harvested and washed with phosphate-buf- RESULTS fered saline. The cell pellets were extracted with 0.5 ml of 50 mM Tris-HC1 pH 7-2, 5 mM dithioand OD~-InhibitingActivity in Rat threitol, O-lmM EDTA and 0-5 per cent Triton X ODC Thymocytes 100 (lysis buffer). The cell lysates were centrifuged The thymus from young rats contained high at 10 000 g for 15 min. ODC activity was assayed in the supernatants by measuring the amount of levels of ODC, in part as a complexed, cryptic form, 14C0, formed during 60 min of incubation at 37°C. releasable by an excess of DFMO-inactivated
245
ORNITHINE DECARBOXYLASE IN RAT THYMOCYTES
ODC added to the thymus extract (Table 1). Treatment with dexamethasone for 5 h caused a marked reduction of free ODC activity (to less than 10 per cent), an appreciable reduction of complexed ODC and the appearance of a remarkable ODC-inhibiting activity. These results are in agreement with a previous report by Peng et aL9 On the other hand, Table 1 also shows that thymocytes obtained from the thymus of untreated animals did not show detectable ODC, either free or complexed, despite the fact that these cells represent the bulk of the cell population of the thymus. However, thymocyte lysates contained appreciable ODC inhibiting activity. This inhibitory activity resembled that of antizyme, in that it was time-independent and nondialysable (not reported). However analysis of the thymocyte antizyme-like activity by high performance gel chromatography on Bio Sil TSK 250 column showed a heterogeneous profile (Figure 1). A major peak was observed at a Mr of about 50 KDa, a value corresponding to that reported previously for the ODC inhibitor partially purified from the thymus of dexamethasone-treated ratsz8 A minor peak corresponding at a Mr of about 25 KDa was also noted, but a large proportion of the inhibiting activity was also observed at higher Mr values (150 KDa and more). The profile of the ODC inhibiting activity resembled that of crude extract of the dexamethasone-treated rat thymus (Figure 1). Incubation of thymocytes in the RPMI 1640 medium supplemented with 10 per cent serum increased the ODC inhibiting activity remarkably up to 6 h (Figure 2) and the activity was still high after 18 h. Omission of serum from the cell medium did not prevent the appearance of the inhibiting activity. Cycloheximide, an inhibitor of protein synthesis, caused an initial stimulation of inhibiting activity, but after 6 h the level of the
158
66
30
20
44 29
17
40
Source
'.
inhibitor was lower with respect to untreated cells. The addition of dexamethasone did not affect the presence and the levels of the inhibitor in thymocytes (Figure 2). It should be noted that complexed or free ODC activity was not detected in dexamethasone- or cycloheximide-treated cells nor in untreated thymocytes, even if the thymocytes were seeded at low density (lo5cells m1-I). Since polyamines are known to induce antizyme in several tissues and cell line^,^,^ the polyamine content of cultured thymocytes was determined. Table 2 shows that putrescine, spermidine and spermine content did not increase in the cells;
Thymus Thymus Thymocytes
None Dex None
Complexed ODC
'
Treatment
ODC-inhibiting activity
(units mg- protein) 1.18 0.32 0.10 & 0.04 n.d.
60
Figure 1. Gel filtration (Bio Sil TSK 250) HPLC of ODCinhibiting activity from rat thymocytes (0) or from the thymus of dexamethasone-treated rats (A). The extracts obtained from cell or thymus homogenates were diluted to a protein concentration of 4 mg m1-l. Then 0.5 ml were chromatographated on a 600 x 7.5 mm Bio Sil TSK 250 column eluted at a flow rate of 0.5 ml min- ' with 50 mM Tris pH 7, 1 mM DTT, 0.1 mM EDTA and 0.3 M NaC1. Fractions of 0.5 ml were collected and 40 p1 were assayed for ODC-inhibition, by using an amount of partially purified ODC giving 2500 dpm(assay)- The position of standard proteins of known Mr (KDa) is indicated.
Table 1. ODC activity, ODC-inhibiting activity and complexed ODC in rat thymus and in cultured rat thymocytes. ODC activity
50
retention time (rnin)
0.62 & 0.21 0.26 & 0.09 n.d.
n.d. 1.31 f 0.20 0.33 & 0.15
Extracts were prepared from thymus of control rats, from thymus of rats treated with 2 mg kg-' dexamethasone for 5 h (Dex), or from isolated thymocytes. Results are mean f S.E. of three determinations. n.d., not detected.
246
C. STEFANELLI ET AL.
thymocytes was accompanied by the complete disappearance of ODC, but did not depend on the polamine content. In the rat thymus the decrease of ODC caused by dexamethasone was accompanied by a concomitant induction of spermidine N’ acetyltransferase a ~ t i v i t y , ’so ~ we have tested spermidine acetylating activity in thymocytes. In isolated cells spermidine acetylation remained unchanged at least for 24 h at a level of about 40 pmol min-’ mg-’ of protein, similar to the activity found in the intact rat thyrnu~.’~
G E 4[
Induction of ODC Actiuity in Rat Thymocytes
0
2
4
6
18
Time (h) Figure 2. Time course of ODC-inhibitory activity in isolated thymocytes. Thymocytes were isolated from the thymuses of control rats and incubated at the cell density of lo7 ml-’ in RPMI 1640 medium supplemented with 10 per cent serum. Samples of untreated thymocytes (e),thymocytes treated with 1 PM dexamethasone (A) and thymocytes treated with 0.1 mM cycloheximide (0)were collected at the times indicated and the ODC-inhibiting activity was assayed in cell extracts.
Table 2. Polyamine content in cultured rat thymocytes.
Time (h)
Polyamine content (nmol (lo8 cells)- I ) Putrescine Spermidine Spermine
0 4
4.6 & 0.8 4.2 f 0.8
8
3.8 0.5 3.1 & 0.4
24
33 f 5 31 k 4 30 & 4 27 f 5
25 f 3 23 f 4 24 & 2 22 & 3
Thymocytes were isolated from thymuses of untreated rats. The cells were then diluted to lo7cells ml- divided in 5 ml aliquots in flasks and incubated at 37°C. Polyamines were measured by HPLC at various times after dilution. Results are mean & S.E. of three flasks.
’,
rather they underwent a slow decline, probably caused by the lack of ODC activity. Furthermore, addition of spermidine (1 mM) did not increase further the antizyme-like activity of thymocytes (not shown). Therefore, these results indicate that the presence of the ODC-inhibiting activity in
The lack of ODC activity in thymocytes, compared to the high levels of ODC in the thymus of the whole rat, suggested that expression of ODC activity in these cells requires the continuous presence of some hormonal or growth factors. In accordance with previous reports,31 addition of Con A, a mitogen known to induce ODC in various lymphoid cells and caused a marked stimulation of ODC activity in thymocytes which peaked after 4-6 h (Figure 3a). Other compounds that can affect lymphocyte responses, such as the phorbol ester PMA, and several hormones, i.e. T3, insulin, glucagon and adrenalin, were all able to induce ODC activity (Figure 3b). However the stimulation was more rapid, but more transient and less marked than that caused by Con A. Actinomycin D, an inhibitor of transcription, paradoxically caused the appearance of ODC activity with a time course similar to that shown by PMA and the hormones (Figure 3C). No free antizyme or ODC-antizyme complex was detected in thymocytes treated with Con A, hormones or actinomycin D. The induction of ODC by Con A, hormones or actinomycin D depended almost completely on the presence of serum in the cell medium (Table 3). Furthermore the presence of cycloheximide (0.2 mM) or spermidine (5 mM) in the medium abolished the inducibility of ODC, indicating that the appearance of ODC activity depended on the translation of its mRNA. In Con A-activated thymocytes, where ODC activity was restored, the enzyme activity decayed with a half-life of about 40 min, after protein synthesis was blocked with cycloheximide (Figure 4). It is interesting to note that ODC turnover occurred at a similar rate in rat thymus of untreated rats in viuo, indicating that isolation of the thymocyte did not affect the ODC-degrading system.
247
ORNITHINE DECARBOXYLASE IN RAT THYMOCYTES 3r
u
n
0
.i
3 f
i i
0 ; 1 0 01 2 4 6 Time (h)
2
0 Time (h)
4
6
Time (h)
Figure 3. Time course of ODC activity in thymocytes stimulated with mitogens and hormones. Thymocytes were isolated and incubated as follows: (a) thymocytes were incubated in the absence (0) or in the presence ( 0 )of Con A (lOpgml-'). (b) Thymocytes were incubated in the presence of 20 ng ml-' PMA (A), 1 p~ T3 (A),10 pg ml-' insulin (U), 10 pg ml- ' glucagon or 5 p~ adrenalin (c) Thymocytes were incubated in the presence of 5 pg ml-' actinomycin D. Results are the means of three separate experiments; S.E. ranged from 4 to 23 per cent.
(m)
(v).
Table 3. Effect of some variables on the induction of ODC in rat thymocytes.
Variable Inducer None Con A (10pgml-') PMA (20ngml-') Actinomycin D ( 5 pg m1- l ) Adrenalin ( 5 PM)
None
ODC activity (units mg- ') Lack of Cycloheximide serum (0.2 rnM)
Spermidine (5 mM)
n.d.
n.d.
n.d.
n.d.
9.8 f 1.7
0.4 f 0.2
n.d.
1.5 f 0.4
0.2 f 0.1
n.d.
n.d.
3.2
0.9
0.8 & 0.2
n.d.
n.d.
2.1 f 0.6
0.2 f 0.1
n.d.
n.d.
0.6
0.3
Thymocytes were suspended at a density of lo7 cellsml-' and treated with ODC inducers in absence and in presence of cycloheximide or spermidine. The cells were incubated 4 h (in presence of Con A) or 2 h (in presence of PMA, actinomycin D and adrenalin) and then harvested for the ODC assay. Results are means f S.E. of three experiments. n.d. not detected.
A major peak with an Mr of about 50 KDa and a shoulder corresponding to about 25 KDa were An interesting finding of the present work is that observed together with peaks eluting at positions isolation of thymocytes from thymus resulted in the corresponding to higher Mr. A somehow similar complete disappearance of the high levels of ODC profile was exhibited by crude extracts of thymus that characterize the thymus of young rats and from dexamethasone-treated rats. It should be notelicited an ODC-inhibiting activity. This activity ed that a Mr of about 50 KDa has been reported resembled antizyme in many respects, but appeared from the purified ODC inhibitor from dexamethaheterogeneous following analysis on gel filtration. sone-treated rat thymus2* and Mr values ranging
DISCUSSION
248
C. STEFANELLI ET AL.
lease of inhibitor bound to macromolecular or subcellular sites3 may be involved together with new synthesis. In isolated thymocytes the levels of ODC and inhibitor varied in an inverse relationship: it is conceivable that withdrawal of hormonal and immunological effectors, which maintain high levels of thymic ODC in the whole animal, causes the disappearance of ODC in cultured, quiescent thymocytes and at the same time allows the conditions for the appearance of the antizyme-like activity. Interestingly lymphoma cells have been reported to express an ODC inhibitor while they remained in a quiescent status, whereas ODC was induced when 0 20 40 60 they were stimulated to proliferate by treatment Time a f t e r cycloheximide (rnin) with p r ~ l a c t i nIn . ~this ~ investigation, addition of a Figure 4. ODC turnover in thymus of control rats ( 0 )or in variety of hormones and effectors that affect the isolated thymocytes treated with Con A for 4 h (0).Cyclohexi- growth and immunological functions of lymphomide was added to intact animals (20mgkg-') or to the cytes positively, restored the ODC activity in thythymocyte medium (0.2 mM) in order to measure the decay of mocytes. A long-lasting and marked enhancement ODC activity in thymus or thymocytes, respectively. in ODC activity was observed following treatment with Con A, as observed by several investigators in lymphoid cells.12,'3*31Th is mitogen has been from 19 KDa to 27 KDa have been found for rat shown to increase remarkably the content of ODC liver a n t i ~ y m e . ~It' is possible that most of the mRNA in bovine lymphocyte^'^ and seems also to heterogeneity observed in crude extracts derives induce a rapid activation (within minutes) of thyfrom the ability of the ODC inhibitor to aggregate, mocyte ODC activity.37 The effects of PMA and despite the presence of a relatively high salt concen- hormones was earlier, less well marked and more tration in the elution buffer. This possibility is transient, possibly as a consequence of a translasupported by the results reported by Matsufuji et tional activation of existing ODC mRNA. A similar d3 who, employing seven different monoclonal mechanism may explain the paradoxical and tranantibodies raised against rat liver antizyme, showed sient ODC stimulation by actinomycin D, an immuno-crossreactivity with antizymes from all the inhibitor of transcription. The induction by actinorat tissues examined, including the spleen of dexa- mycin D was prevented by cycloheximide, suggestmethasone-treated rats. Furthermore, similar-sized ing that this chemical increases ODC synthesis. antizyme mRNAs were found in various rat tissues, 'Superinduction' of ODC by actinomycin D, attriincluding the thymus." The intracellular content buted to increased translational efficiency, has been of antizyme is thought to depend upon the levels of shown in lipopolysaccharide-stimulated rat spleen polyamines, which, acting post-transcriptionally, by end^,'^ and in ELD cells by Wallon et ~ f . , ~ * can induce antizyme in several tissues or who detected increased ODC synthesis in the presce11s.3,4,0,33-35 H owever, in the present investiga- ence of an unmodified content of ODC mRNA. The tion the rise of ODC-inhibiting activity in cultured induction of ODC by actinomycin D may also thymocytes occurred in the absence of any increase explain the results reported by Brand3' who in polyamine content. Thus effectors other than showed that actinomycin D could not inhibit ODC polyamines may control the level of antizyme-like induction by IL-2. In conclusion these results indicate that expresinhibitors. The possibility of a direct involvement of hormones in the modulation of the levels of ODC sion of ODC activity in thymic lymphocytes reinhibitors also emerges from the analysis of the quires the stimulation with hormones or mitogens, polyamine content of spleenz9 and thymusg of the normal components of serum being necessary dexamethasone-treated rats. It should also be not- but not sufficient to maintain the high ODC actied that cycloheximide initially increased the ODC- vity typical of thymus in the intact animal. An inhibiting activity in thymocytes and only subse- important role in modulating the cellular levels of quently blocked it. It may be speculated that re- ODC may be played by an antizyme-like inhibitor,
249
ORNITHINE DECARBOXYLASE IN RAT THYMOCYTES
which is present in a cryptic form in unstimulated 2. thymus and accumulates in isolated thymocytes to a large extent. In this regard, the fact that isolated thymocytes express either ODC or inhibitor activi- 3. ty, whereas the ODC-inhibitor complex does not accumulate appreciably, may be consistent with the hypothesis that complexed ODC undergoes a rapid degradati~n.~." 14.
ACKNOWLEDGEMENTS This research was supported by grants from M.U.R.S.T. and from C.N.R. Target Project on Aging (publication code 921206).
15.
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dexamethasone on the activity and expression of ornithine decarboxylase in rat liver and thymus. Biochim. Biophys. Acta, 950,229-233. Bishop, P. B., Young, J., Peng, T. and Richards, J. F. (1985). An inhibitor of ornithine decarboxylase in the thymus and spleen of dexamethasone-treated rats. Biochem. J., 226, 105- 1 12. Stefanelli, C., Flamigni, F., Carati, D., Rossoni, C. and Caldarera, C. M. (1987). Effects of dexamethasone on spermidine N'-acetyltransferase and ornithine decarboxylase activities in rat spleen. Biochim. Biophys. Acta, 930, 79-86. Stefanelli,C., Carati, D. and Rossoni, C. (1986). Separation of N' and N*-acetylspermidine isomers by reversedphased column liquid chromatography after derivatization with dansyl chloride. J . Chromatogr., 375,49-55. Brand, K. (1987). Role of ornithine decarboxylase on glycolytic enzyme induction during thymocytes proliferation. J. Biol. Chem., 262, 15232-15235. Kitani, T. and Fujsawa, H. (1984). Purification and some properties of a protein inhibitor (antizyme) of ornithine decarboxylase from rat liver. J. Biol. Chem., 259, 10036-10040. Matsufuji, S., Kanamoto, R., Murakami, Y. and Hayashi, S. (1990). Monoclonal antibody studies on the properties and regulation of murine ornithine decarboxylase antizymes. J. Biochem., 107, 87-91.
34. Brosnan, M. E., Farrell, R., Wilanski, H. and Williamson, D. H. (1983). Effects of starvation and refeeding of polyamine concentrations and ornithine decarboxylase antizyme in mammary gland of lactating rats. Biochem. J., 212, 149-153. 35. Murakami, Y., Nishiyama, M. and Hayashi, S. (1989). Involvement of antizyme in stabilization of ornithine decarboxylase caused by inhibitors of polyamine synthesis. Eur. J. Biochem., 180, 181-184. 36. Richards, J. F., Bishop, P. B., Peng, T., Beer, C. T. and Gout, P. W. (1986). An inhibitor of ornithine decarboxylase in lactogen-deprived Nb2 node rat lymphoma cells. Biochim. Biophys. Acta, 883, 542-551. 37. Valette, L., Fonlupt, P., Prigent, A. F., Macoviski, 0. and Lagarde, M. (1990). Early induction of ornithine decarboxylase occurs simultaneously with inositol phosphate accumulation in concanavalin-A stimulated rat thymocytes. Immunol. Lett., 26, 233-231. 38. Wallon, M. V., Persson, L. and Heby, 0. (1990). Superinduction of ornithine decarboxylase (ODC) by actinomycin D is due to stimulation of ODC mRNA translation. FEBS Lett., 268, 161-164.
Received 27 February 1992 Accepted 31 March 1992
10: 243-250 (1992)
Ornithine Decarboxylase and Ornithine Decarboxylaseinhibiting Activity in Rat Thymocytes CLAUDIO STEFANELLI, CARMEN ROSSONI, FABRIZIA FERRARI, FLAVIO FLAMIGNI AND CLAUDIO M. CALDARERA Department of Biochemistry ‘G. Moruzzi’, University of Bologna, 40126 Bologna, Italy
Isolation of thymocytes from rat thymus resulted in the disappearance of the high activity of ornithine decarboxylase (ODC) that characterizes the thymus of young rats, together with the appearance of an antizyme-like ODC inhibiting activity, which showed a chromatographic profile that resembled that of dexamethasone-treated rat thymus. Omission of serum or addition of dexamethasone or spermidine did not affect appreciably the extent of the antizymelike activity. On the other hand, a variety of hormonal effectors, i.e. insulin, glucagon, adrenalin and T3, as well as the phorbol ester, PMA or the mitogen, concanavalin A (Con A) induced ODC activity in cultured thymocytes together with the disappearance of the antizyme-like activity. A paradoxical, transient induction of ODC was caused by the transcriptional inhibitor, actinomycin D. Complexed ODC was detected in rat thymus, but not in thymocytes, either quiescent or stimulated by mitogens. These results indicate that thymic lymphocytes can express either ODC activity or its inhibitor depending on the hormonal and proliferative status of the cells. KEY
WORDS--0rnithine decarboxylase; antizyme; thymocytes; polyamines.
ABBREVIATIONS-ODC, ornithine decarboxylase; Con A, concanavalin A; HPLC, high performance liquid chromatography; PMA, phorbol- 12-myristate-13-acetate; T3, 3,5,3’-triiodothyronine.
INTRODUCTION The importance of the polyamines, putrescine, The relevance of each particular regulatory spermidine and spermine, in cell growth and differ- mechanism of ODC appears to depend on the entiation is reflected by the complexity of the specific stimuli and the cell type. In lymphoid cells, regulation of ornithine decarboxylase (ODC), the marked increases in ODC levels are observed rate limiting enzyme in polyamine biosynthesis. following activation with mitogens, antigens or Transcriptional, translational and post-transla- phorbol esters.”-16 Active ODC and polyamines tional control of ODC has been described,’,’ as are required for the interleukin-2-dependent well as its interaction with a specific protein inhibi- growth and alloantigen activation of cytolytic tor, named a n t i ~ y m eAlthough .~ antizyme was ini- T-lymphocyte~’~~” and are involved in the down tially discovered in tissues or cells exposed to high regulation of the interleukin-2 production by T concentrations of polyamines: antizyme mRNA cells in EL-4 lymphoma cells.I8 and ODC-antizyme complex were subsequently Changes in ODC activity also occur in lymphoid found to be present constitutively in several rodent organs, such as the thymus and spleen, in relation Furthermore, it has been proposed that to their trophic and functional state. Thus, ODC ODC complexed to antizyme is not only catalyti- activity is very high in the thymus of young anically inactive, but also rapidly degraded.” mals, but declines with age, as the organ undergoes i n v o l ~ t i o n . ’ODC ~ activity is increased in lymphoid organs stimulated not only by mitogens but also by hormones, such as insulin, catecholamines, Conversely, Address for correspondence: Dr Claudio Stehnelli, Diparti- growth hormone and pr~lactin.’~-’~ mento di Biochimica, via Irnerio 48, 40126 Bologna, Italy. ODC levels decrease dramatically in the thymus 0263-6484/92/340243-08%09.00 01992 by John Wiley & Sons, Ltd.
244
C. STEFANELLI ET AL.
and spleen of animals injected with dexamethasone, preceding by several hours the cytolytic effect of the glucocorticoid.26 In particular, in the thymus of rats treated with dexamethasone, the reduction in the content of ODC mRNA could account only partially for the marked decrease in ODC activity.” However, dexamethasone caused the appearance of an antizyme-like inhibitor of ODC in thymus and spleen extract^.^^*^^ In the present study, we have investigated conditions and stimuli leading to the expression of ODC and antizyme activity in isolated thymic lymphocytes (thymocytes).
One unit of ODC activity is defined as the amount releasing 1 nmol CO, h-’. For the determination of ODC inhibitory activity6 cell extracts were tested for their ability to inhibit a fixed amount of ODC from rat thymus that had been partially purified by ammonium sulphate precipitation followed by high performance ion exchange chromatography on a Bio-Gel TSK-DEAE-5-PW column and gel filtration on a Bio Sil TSK-250 column. One unit corresponds to the amount inhibiting 1 unit of ODC activity. The amount of complexed ODC was measured by assaying in 40 pl of extract the enzyme activity releasable by an excess of rat thymus ODC inactivated by a-difluoromethyl ornithine (DFMO) as MATERIALS AND METHODS previously described.6 High performance gel filtration chromatography Chemicals of ODC-inhibiting activity was performed on a The chemicals for preparation of culture media TSK-250 600 x 7.5 mm column calibrated with were obtained from Gibco Laboratories. Biochemi- standard proteins of known Mr. For this determicals were from Sigma Chemical Co. and other nation about lo8 cells were lysed in 0-8ml of lysis buffer. After centrifugation, 0.5 ml of crude extract laboratory chemicals were products of E. Merck. was injected and eluted with 50 mM Tris-HC1 pH 7.0 containing 0.3 M NaCl, 0.1 mM EDTA and Animals and Cell Cultures 1 mM dithiothreitol at a flow rate of 0.5 ml minFractions of 0.5 ml were collected and 4Opl of Male Wistar rats were used. Thymocytes were every fraction were tested for ODC-inhibitory acobtained from the thymuses of 4 to 10 week-old animals. Thymus glands were removed, washed in tivity, using a fixed amount of purified ODC giving phosphate-buffered saline and immersed in a stan- 2500 dpm (assay)- in the absence of inhibitors. dard medium containing RPMl 1640 with 10 per Rat thymus extracts were prepared as previously cent fetal bovine serum, 2 mM glutamine, 25 mM de~cribed.’~ HEPES, penicillin and streptomycin (100 units ml-l). Thymuses were chopped into small Other Assays pieces and disaggregated. The cell suspension was For the determination of polyamines the pelleted filtered and centrifuged at 800 g for 5 min. The cells were resuspended in 0.5 ml of chilled 0.3 M pelleted cells were resuspended in 2 ml of standard PCA and homogenized by repeated freezing and medium, counted and diluted to lo7 cellsml-’. thawing. After centrifugation polyamines were Cell suspensions (5 ml) were incubated in sterile flasks in a humidified atmosphere of 5 per cent CO, measured in acid extracts by HPLC.30 Spermidine acetyltransferase activity was measured in the same in air at 37°C. extracts used for the ODC assay by measuring the incorporation of labelled acetyl coenzyme A into ODC and ODC-Inhibitory Activities per mi dine.'^
’.
At the time indicated after treatment, the cells were harvested and washed with phosphate-buf- RESULTS fered saline. The cell pellets were extracted with 0.5 ml of 50 mM Tris-HC1 pH 7-2, 5 mM dithioand OD~-InhibitingActivity in Rat threitol, O-lmM EDTA and 0-5 per cent Triton X ODC Thymocytes 100 (lysis buffer). The cell lysates were centrifuged The thymus from young rats contained high at 10 000 g for 15 min. ODC activity was assayed in the supernatants by measuring the amount of levels of ODC, in part as a complexed, cryptic form, 14C0, formed during 60 min of incubation at 37°C. releasable by an excess of DFMO-inactivated
245
ORNITHINE DECARBOXYLASE IN RAT THYMOCYTES
ODC added to the thymus extract (Table 1). Treatment with dexamethasone for 5 h caused a marked reduction of free ODC activity (to less than 10 per cent), an appreciable reduction of complexed ODC and the appearance of a remarkable ODC-inhibiting activity. These results are in agreement with a previous report by Peng et aL9 On the other hand, Table 1 also shows that thymocytes obtained from the thymus of untreated animals did not show detectable ODC, either free or complexed, despite the fact that these cells represent the bulk of the cell population of the thymus. However, thymocyte lysates contained appreciable ODC inhibiting activity. This inhibitory activity resembled that of antizyme, in that it was time-independent and nondialysable (not reported). However analysis of the thymocyte antizyme-like activity by high performance gel chromatography on Bio Sil TSK 250 column showed a heterogeneous profile (Figure 1). A major peak was observed at a Mr of about 50 KDa, a value corresponding to that reported previously for the ODC inhibitor partially purified from the thymus of dexamethasone-treated ratsz8 A minor peak corresponding at a Mr of about 25 KDa was also noted, but a large proportion of the inhibiting activity was also observed at higher Mr values (150 KDa and more). The profile of the ODC inhibiting activity resembled that of crude extract of the dexamethasone-treated rat thymus (Figure 1). Incubation of thymocytes in the RPMI 1640 medium supplemented with 10 per cent serum increased the ODC inhibiting activity remarkably up to 6 h (Figure 2) and the activity was still high after 18 h. Omission of serum from the cell medium did not prevent the appearance of the inhibiting activity. Cycloheximide, an inhibitor of protein synthesis, caused an initial stimulation of inhibiting activity, but after 6 h the level of the
158
66
30
20
44 29
17
40
Source
'.
inhibitor was lower with respect to untreated cells. The addition of dexamethasone did not affect the presence and the levels of the inhibitor in thymocytes (Figure 2). It should be noted that complexed or free ODC activity was not detected in dexamethasone- or cycloheximide-treated cells nor in untreated thymocytes, even if the thymocytes were seeded at low density (lo5cells m1-I). Since polyamines are known to induce antizyme in several tissues and cell line^,^,^ the polyamine content of cultured thymocytes was determined. Table 2 shows that putrescine, spermidine and spermine content did not increase in the cells;
Thymus Thymus Thymocytes
None Dex None
Complexed ODC
'
Treatment
ODC-inhibiting activity
(units mg- protein) 1.18 0.32 0.10 & 0.04 n.d.
60
Figure 1. Gel filtration (Bio Sil TSK 250) HPLC of ODCinhibiting activity from rat thymocytes (0) or from the thymus of dexamethasone-treated rats (A). The extracts obtained from cell or thymus homogenates were diluted to a protein concentration of 4 mg m1-l. Then 0.5 ml were chromatographated on a 600 x 7.5 mm Bio Sil TSK 250 column eluted at a flow rate of 0.5 ml min- ' with 50 mM Tris pH 7, 1 mM DTT, 0.1 mM EDTA and 0.3 M NaC1. Fractions of 0.5 ml were collected and 40 p1 were assayed for ODC-inhibition, by using an amount of partially purified ODC giving 2500 dpm(assay)- The position of standard proteins of known Mr (KDa) is indicated.
Table 1. ODC activity, ODC-inhibiting activity and complexed ODC in rat thymus and in cultured rat thymocytes. ODC activity
50
retention time (rnin)
0.62 & 0.21 0.26 & 0.09 n.d.
n.d. 1.31 f 0.20 0.33 & 0.15
Extracts were prepared from thymus of control rats, from thymus of rats treated with 2 mg kg-' dexamethasone for 5 h (Dex), or from isolated thymocytes. Results are mean f S.E. of three determinations. n.d., not detected.
246
C. STEFANELLI ET AL.
thymocytes was accompanied by the complete disappearance of ODC, but did not depend on the polamine content. In the rat thymus the decrease of ODC caused by dexamethasone was accompanied by a concomitant induction of spermidine N’ acetyltransferase a ~ t i v i t y , ’so ~ we have tested spermidine acetylating activity in thymocytes. In isolated cells spermidine acetylation remained unchanged at least for 24 h at a level of about 40 pmol min-’ mg-’ of protein, similar to the activity found in the intact rat thyrnu~.’~
G E 4[
Induction of ODC Actiuity in Rat Thymocytes
0
2
4
6
18
Time (h) Figure 2. Time course of ODC-inhibitory activity in isolated thymocytes. Thymocytes were isolated from the thymuses of control rats and incubated at the cell density of lo7 ml-’ in RPMI 1640 medium supplemented with 10 per cent serum. Samples of untreated thymocytes (e),thymocytes treated with 1 PM dexamethasone (A) and thymocytes treated with 0.1 mM cycloheximide (0)were collected at the times indicated and the ODC-inhibiting activity was assayed in cell extracts.
Table 2. Polyamine content in cultured rat thymocytes.
Time (h)
Polyamine content (nmol (lo8 cells)- I ) Putrescine Spermidine Spermine
0 4
4.6 & 0.8 4.2 f 0.8
8
3.8 0.5 3.1 & 0.4
24
33 f 5 31 k 4 30 & 4 27 f 5
25 f 3 23 f 4 24 & 2 22 & 3
Thymocytes were isolated from thymuses of untreated rats. The cells were then diluted to lo7cells ml- divided in 5 ml aliquots in flasks and incubated at 37°C. Polyamines were measured by HPLC at various times after dilution. Results are mean & S.E. of three flasks.
’,
rather they underwent a slow decline, probably caused by the lack of ODC activity. Furthermore, addition of spermidine (1 mM) did not increase further the antizyme-like activity of thymocytes (not shown). Therefore, these results indicate that the presence of the ODC-inhibiting activity in
The lack of ODC activity in thymocytes, compared to the high levels of ODC in the thymus of the whole rat, suggested that expression of ODC activity in these cells requires the continuous presence of some hormonal or growth factors. In accordance with previous reports,31 addition of Con A, a mitogen known to induce ODC in various lymphoid cells and caused a marked stimulation of ODC activity in thymocytes which peaked after 4-6 h (Figure 3a). Other compounds that can affect lymphocyte responses, such as the phorbol ester PMA, and several hormones, i.e. T3, insulin, glucagon and adrenalin, were all able to induce ODC activity (Figure 3b). However the stimulation was more rapid, but more transient and less marked than that caused by Con A. Actinomycin D, an inhibitor of transcription, paradoxically caused the appearance of ODC activity with a time course similar to that shown by PMA and the hormones (Figure 3C). No free antizyme or ODC-antizyme complex was detected in thymocytes treated with Con A, hormones or actinomycin D. The induction of ODC by Con A, hormones or actinomycin D depended almost completely on the presence of serum in the cell medium (Table 3). Furthermore the presence of cycloheximide (0.2 mM) or spermidine (5 mM) in the medium abolished the inducibility of ODC, indicating that the appearance of ODC activity depended on the translation of its mRNA. In Con A-activated thymocytes, where ODC activity was restored, the enzyme activity decayed with a half-life of about 40 min, after protein synthesis was blocked with cycloheximide (Figure 4). It is interesting to note that ODC turnover occurred at a similar rate in rat thymus of untreated rats in viuo, indicating that isolation of the thymocyte did not affect the ODC-degrading system.
247
ORNITHINE DECARBOXYLASE IN RAT THYMOCYTES 3r
u
n
0
.i
3 f
i i
0 ; 1 0 01 2 4 6 Time (h)
2
0 Time (h)
4
6
Time (h)
Figure 3. Time course of ODC activity in thymocytes stimulated with mitogens and hormones. Thymocytes were isolated and incubated as follows: (a) thymocytes were incubated in the absence (0) or in the presence ( 0 )of Con A (lOpgml-'). (b) Thymocytes were incubated in the presence of 20 ng ml-' PMA (A), 1 p~ T3 (A),10 pg ml-' insulin (U), 10 pg ml- ' glucagon or 5 p~ adrenalin (c) Thymocytes were incubated in the presence of 5 pg ml-' actinomycin D. Results are the means of three separate experiments; S.E. ranged from 4 to 23 per cent.
(m)
(v).
Table 3. Effect of some variables on the induction of ODC in rat thymocytes.
Variable Inducer None Con A (10pgml-') PMA (20ngml-') Actinomycin D ( 5 pg m1- l ) Adrenalin ( 5 PM)
None
ODC activity (units mg- ') Lack of Cycloheximide serum (0.2 rnM)
Spermidine (5 mM)
n.d.
n.d.
n.d.
n.d.
9.8 f 1.7
0.4 f 0.2
n.d.
1.5 f 0.4
0.2 f 0.1
n.d.
n.d.
3.2
0.9
0.8 & 0.2
n.d.
n.d.
2.1 f 0.6
0.2 f 0.1
n.d.
n.d.
0.6
0.3
Thymocytes were suspended at a density of lo7 cellsml-' and treated with ODC inducers in absence and in presence of cycloheximide or spermidine. The cells were incubated 4 h (in presence of Con A) or 2 h (in presence of PMA, actinomycin D and adrenalin) and then harvested for the ODC assay. Results are means f S.E. of three experiments. n.d. not detected.
A major peak with an Mr of about 50 KDa and a shoulder corresponding to about 25 KDa were An interesting finding of the present work is that observed together with peaks eluting at positions isolation of thymocytes from thymus resulted in the corresponding to higher Mr. A somehow similar complete disappearance of the high levels of ODC profile was exhibited by crude extracts of thymus that characterize the thymus of young rats and from dexamethasone-treated rats. It should be notelicited an ODC-inhibiting activity. This activity ed that a Mr of about 50 KDa has been reported resembled antizyme in many respects, but appeared from the purified ODC inhibitor from dexamethaheterogeneous following analysis on gel filtration. sone-treated rat thymus2* and Mr values ranging
DISCUSSION
248
C. STEFANELLI ET AL.
lease of inhibitor bound to macromolecular or subcellular sites3 may be involved together with new synthesis. In isolated thymocytes the levels of ODC and inhibitor varied in an inverse relationship: it is conceivable that withdrawal of hormonal and immunological effectors, which maintain high levels of thymic ODC in the whole animal, causes the disappearance of ODC in cultured, quiescent thymocytes and at the same time allows the conditions for the appearance of the antizyme-like activity. Interestingly lymphoma cells have been reported to express an ODC inhibitor while they remained in a quiescent status, whereas ODC was induced when 0 20 40 60 they were stimulated to proliferate by treatment Time a f t e r cycloheximide (rnin) with p r ~ l a c t i nIn . ~this ~ investigation, addition of a Figure 4. ODC turnover in thymus of control rats ( 0 )or in variety of hormones and effectors that affect the isolated thymocytes treated with Con A for 4 h (0).Cyclohexi- growth and immunological functions of lymphomide was added to intact animals (20mgkg-') or to the cytes positively, restored the ODC activity in thythymocyte medium (0.2 mM) in order to measure the decay of mocytes. A long-lasting and marked enhancement ODC activity in thymus or thymocytes, respectively. in ODC activity was observed following treatment with Con A, as observed by several investigators in lymphoid cells.12,'3*31Th is mitogen has been from 19 KDa to 27 KDa have been found for rat shown to increase remarkably the content of ODC liver a n t i ~ y m e . ~It' is possible that most of the mRNA in bovine lymphocyte^'^ and seems also to heterogeneity observed in crude extracts derives induce a rapid activation (within minutes) of thyfrom the ability of the ODC inhibitor to aggregate, mocyte ODC activity.37 The effects of PMA and despite the presence of a relatively high salt concen- hormones was earlier, less well marked and more tration in the elution buffer. This possibility is transient, possibly as a consequence of a translasupported by the results reported by Matsufuji et tional activation of existing ODC mRNA. A similar d3 who, employing seven different monoclonal mechanism may explain the paradoxical and tranantibodies raised against rat liver antizyme, showed sient ODC stimulation by actinomycin D, an immuno-crossreactivity with antizymes from all the inhibitor of transcription. The induction by actinorat tissues examined, including the spleen of dexa- mycin D was prevented by cycloheximide, suggestmethasone-treated rats. Furthermore, similar-sized ing that this chemical increases ODC synthesis. antizyme mRNAs were found in various rat tissues, 'Superinduction' of ODC by actinomycin D, attriincluding the thymus." The intracellular content buted to increased translational efficiency, has been of antizyme is thought to depend upon the levels of shown in lipopolysaccharide-stimulated rat spleen polyamines, which, acting post-transcriptionally, by end^,'^ and in ELD cells by Wallon et ~ f . , ~ * can induce antizyme in several tissues or who detected increased ODC synthesis in the presce11s.3,4,0,33-35 H owever, in the present investiga- ence of an unmodified content of ODC mRNA. The tion the rise of ODC-inhibiting activity in cultured induction of ODC by actinomycin D may also thymocytes occurred in the absence of any increase explain the results reported by Brand3' who in polyamine content. Thus effectors other than showed that actinomycin D could not inhibit ODC polyamines may control the level of antizyme-like induction by IL-2. In conclusion these results indicate that expresinhibitors. The possibility of a direct involvement of hormones in the modulation of the levels of ODC sion of ODC activity in thymic lymphocytes reinhibitors also emerges from the analysis of the quires the stimulation with hormones or mitogens, polyamine content of spleenz9 and thymusg of the normal components of serum being necessary dexamethasone-treated rats. It should also be not- but not sufficient to maintain the high ODC actied that cycloheximide initially increased the ODC- vity typical of thymus in the intact animal. An inhibiting activity in thymocytes and only subse- important role in modulating the cellular levels of quently blocked it. It may be speculated that re- ODC may be played by an antizyme-like inhibitor,
249
ORNITHINE DECARBOXYLASE IN RAT THYMOCYTES
which is present in a cryptic form in unstimulated 2. thymus and accumulates in isolated thymocytes to a large extent. In this regard, the fact that isolated thymocytes express either ODC or inhibitor activi- 3. ty, whereas the ODC-inhibitor complex does not accumulate appreciably, may be consistent with the hypothesis that complexed ODC undergoes a rapid degradati~n.~." 14.
ACKNOWLEDGEMENTS This research was supported by grants from M.U.R.S.T. and from C.N.R. Target Project on Aging (publication code 921206).
15.
16,
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Received 27 February 1992 Accepted 31 March 1992
