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Polyamines affect growth of cultured rat cerebellar neurons in different sera
0736-5748/86$03.(XI+(I.IRI PergamonJournalsLtd. (~) 1986ISDN
Int. J. Devl. Neuroscience. Vol. 4, No. 3, pp. 195-2¢18,1986.
Printed in Great Britain.
POLYAMINES AFFECT GROWTH OF C U L T U R E D R A T C E R E B E L L A R N E U R O N S IN DIFFERENT SERA GAD M. GILAD and VARDA H. GILAD The Center for Neuroscience and Behavioral Research and The Department of Isotope Research, The Weizmann Institute of Science, 76100 Rehovot, Israel (Accepted 4 December 1985) A b s t r a c t - - T h e study examines the effects of polyamines on growth of cultured neurons from 6-day-old
rat cerebellar cortex, by means of: (a) irreversible inhibition of ornithine decarboxylase activity with a-difluoromethylornithine, and (b) treatment with the exogenous diamine putrescine and the polyamines spermidine and spermine, in the presence of sera from different sources. Inhibition of ornithine decarboxylase activity starting at plating time led after 24 hr to a partial inhibition of cell aggregation with a drastic (90%) inhibition of neurite formation. However, after 48 hr of enzyme inhibition aggregation and neurite formation increased to approach the 24 hr control values and eventually cultures fully recovered. Polyamines added at plating time in the presence of fetal calf serum led to a permanent dose-dependent inhibition of aggregation and neurite formation, spermine being effective at lower doses (spermine < spermidine ,~ putrescine). Adhesion of cells to polylysine coated surfaces was not affected by polyamines. Recovery from the cytostatic polyamine effect was observed after washing and addition of fresh medium. Prevention of the effect was achieved in the presence of aminoguanidine, an inhibitor of diamine and polyamine oxidases. The preventive effect of aminoguanidine was dose polyamine-dependent, with higher aminoguanidine concentrations needed to prevent the spermine effect (spermine->spermidine > putrescine). The polyamine effects were observed in the presence of fetal calf, heat-inactivated fetal calf and human sera, but not with rat serum. Addition of polyamines to 24-hr-old cultured neurons, in the presence of fetal calf serum, led 12 hr later to cell death. This lethal effect could be inhibited by aminoguanidine. We conclude: (a) irreversible inhibition of ornithine decarboxylase activity delays but does not prevent neuronal growth in culture; (b) oxidation products of extracellular polyamines inhibit cell aggregation and neurite formation of cultured neuroblasts, and have lethal effects on growing neurons in culture, and (c) different pharmacological effects of polyamines can be expected in different species. Key words: Polyamines, Cerebellar cultures, Sera.
T h e b i o g e n i c p o l y a m i n e s ( P A ) s p e r m i d i n e a n d s p e r m i n e , a n d their d i a m i n e p r e c u r s o r p u t r e s c i n e , have b e e n s h o w n to be p r e r e q u i s i t e s for o p t i m a l cellular g r o w t h . 1°'12"24"35"42 P o l y a m i n e s were d e m o n s t r a t e d to affect v a r i o u s r e g u l a t o r y m o l e c u l a r m e c h a n i s m s i n c l u d i n g those of D N A , R N A a n d p r o t e i n synthesis. 1°'24"42 In the b r a i n , P A b i o s y n t h e s i s has b e e n s h o w n to increase rapidly prior to o n s e t of growth. 4"36 It has b e e n r e c e n t l y d e m o n s t r a t e d that selective i n h i b i t i o n of o r n i t h i n e d e c a r b o x y l a s e ( O D C ) activity, the e n z y m e catalyzing the first step in P A biosynthesis, with p u t r e s c i n e a n a l o g s causes i n h i b i t i o n of n e u r i t e f o r m a t i o n by c u l t u r e d chick n e u r o b l a s t s . 39 This growth i n h i b i t i o n can be r e v e r s e d by p u t r e s c i n e i n d i c a t i n g i m p o r t a n t role for P A in n e u r o n a l d i f f e r e n t i a t i o n . This r e c e n t s t u d y indicates that the growth characteristics of n o r m a l n e u r o n s m a y differ from the growth of n e o p l a s t i c p h a e o c h r o m o c y t o m a cells w h e r e i n h i b i t i o n of O D C activity with an a n a l o g of o r n i t h i n e , did n o t affect n e u r i t e outgrowth.19 Paradoxically, h o w e v e r , while i n t r a c e l l u l a r P A are essential for o p t i m a l cell growth, e x o g e n o u s P A can e v o k e p o t e n t s u p p r e s s i o n of cell p r o l i f e r a t i o n a n d cytotoxicity w h e n a d d e d to cell cultures in the p r e s e n c e of r u m i n a n t sera. 5"2°'22 This adverse effect of P A is m a i n l y d u e to the oxidative d e a m i n a t i o n of P A a n d the f o r m a t i o n of a m i n o a l d e h y d e s by p o l y a m i n e oxidase p r e s e n t in these sera.3,9,23,33.43 W e have r e c e n t l y d e m o n s t r a t e d that O D C activity is e l e v a t e d in rat s y m p a t h e t i c n e u r o n s d u r i n g the p e r i n a t a l p e r i o d 15 w h e n n e u r o n a l cell division e n d s a n d n a t u r a l l y o c c u r r i n g n e u r o n a l cell d e a t h b e g i n s ; 21 a p e r i o d which coincides with late stages of growth of t e r m i n a l axons within target tissues. 7 T h e s e findings have suggested that there is an increased d e m a n d for P A d u r i n g that p e r i o d a n d this is reflected by e n h a n c e d P A biosynthesis. B a s e d o n this r a t i o n a l e we have Abbreviations: Ag, aminoguanidine; Ara-c, cytosine arabinoside; DFMO, ct-difluoromethylornithine; ODC, ornithine deearboxylase; PA. polyamines; Put, putrescine, Spd, spermidine; Spin, spermine. DN 4:3-A
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treated rats with exogenous PA for a limited postnatal period and demonstrated that it can lead to an increased number of sympathetic neurons.l~ It is unknown whether PA exert their in vivo effect directly on the neurons themselves, or indirectly through interaction with other cells which in turn exert beneficial effect on the developing neurons. Tissue culture studies may clarify some of these questions. Indeed, the relative ease with which the development of rat cerebellar neuroblasts in cultures can be studied, 28"3°'44together with the indication of the possible involvement of PA in the differentiation of cerebellar interneurons, j4"~617 make such cultures an attractive experimental model for studies of PA pharmacology. In the present study therefore we examined the role of PA in growth of cultured cerebellar neurons using two approaches: (1) inhibition of PA biosynthesis in cultured neurons with e~-difluoromethylornithine ( D F M O ) , an enzyme-activated irreversible inhibitor of O D E 31 and (2) cultivation of neurons with media containing exogenous PA in the presence of: (a) aminoguanidine, an inhibitor of diamine oxidase 4° and, at higher concentrations, of polyamine oxidase,ll'38 and (b) sera from different species. EXPERIMENTAL PROCEDURES
Materials Putrescine dihydrochloride, spermidine trihydrochloride, spermine tetrahydrochloride, aminoguanidine bicarbonate, cytosine arabinoside hydrochloride (Ara-C) and poly-L-iysine hydrobromide (mol. wt 30,000-70,000) were all from Sigma Chemical Co., St. Louis, MO, U.S.A. Ornithine DL-[I-t4C] (sp. act. 49 mCi/mmol) and thymidine [methyl-3H] (sp. act. 6.7 Ci/ mol) were from New England Nuclear, Dreieich, West Germany. Dulbecco's modified Eagle medium and fetal calf serum were from biological Industries Beth H a E m e k , Israel; miramycin (antibiotic) was from Teva, Jerusalem, Israel. DL-et-Difluoromethylornithine ( M D L 71782), was a generous gift from Centre de Recherche Merell International subsidiary of the Dow Chemical Company, Strasbourg, France. Sera Rat trunk blood was collected after decapitation. Human blood was collected by venipuncture. Blood samples were allowed to clot and the sera collected and sterilized by membrane filtration. Sera were heat-inactivated at 57°C for 30 min. Cell culture The brain was rapidly removed from 6-day-old rat pups after decapitation and the cerebellum dissected ~7 and transferred aseptically to a 15 ml sterile plastic conical tube containing 3 ml of Dulbecco's modified Eagle medium containing 10% serum, 2 mM glutamine and 0.1% miramycin. Six to 8 cerebella were pooled together and prepared for culture by adapting the method of Yavin and Yavin. 46 The cerebellum was mechanically dissociated after 15-20 passages through a sterile 13 Ga. 10 cm long stainless steel needle connected through a nylon sieve (48 Ixm pore size) to a 5 ml sterile syringe. The dissociated cerebellum was allowed to settle for a few minutes and the supernatant containing the dissociated cells was collected and centrifuged at 200 g for 5 min. The pellet was resuspended in fresh medium, the cells were counted in a haemocytometer and their viability determined by the Trypan Blue exclusion test. The dissociated cells were cultured (400/m 2) on polylysine coated 46 plastic Petri dishes, or glass coverslips situated in 35 mm plastic Petri dishes. The cultures were incubated at 37°C in an atmosphere of 5% CO2 in air and 100% humidity. Drugs or drug combinations were either added from the beginning, or 24 hr after the beginning of cultures. Cultures were regularly examined in an inverted microscope and photographed with phase contrast microscopy. Thymidine incorporation Medium containing [3H]thymidine, 0.6 I~Ci/dish, was added at plating time or 24 hr after plating. Twenty four hours after thymidine addition cultures were washed twice with fresh
Polyamines and growth of cultured neurons
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medium, the cells scraped off, transferred to counting vials and radioactivity was measured by scintillation spectrometry.
Biochemical assays Cells were scraped off the culture dish and homogenized in 100 mM Tris-HCI buffer (pH 7.4) containing 7.5 mM dithiothreitol, 6 mM ethylenediamine tetraacetic acid (EDTA) and 40 IxM pyridoxal-5'-phosphate. ODC activity was assayed as previously described.'3 Protein concentrations were determined according to the method of Lowry et ai.29 RESULTS
Effects of DFMO ODC activity was lower at the beginning of culture than in age matched cerebellar homogenates (Fig. 1). However, the enzyme activity was transiently elevated with a peak at 24 hr. The activity was still high at 2 days when about 93% of neurons had already extended neurites (Fig. 4), and when thymidine incorporation was low (Fig. 8). By 3 days ODC activity declined to very low levels (Fig. 1).
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Fig. 1. Time-course of changes in ODC activity in cultures of cerebellar cells containing fetal calf serum in the absence (control, solid circle) or in the presence of 5 mM DFMO (open circle). The activity in the homogenate of 6-day-old cerebellum is presented (original homogenate, solid circle). Each point represents the mean ( - S . E . M . ) value of 3 separate experiments with 5 separate determinations (culture dishes) each.
The continuous presence of 5 mM DFMO, from the start, in culture media containing fetal calf serum, completely inhibited ODC activity for the 3 days duration of the experiment (Fig. 1). Morphologically, DFMO led to about 24 hr delay in cell aggregation and neurite formation (Figs 2-4).
Effects of early addition PA The continuous presence of putrescine or PA, from the start, in culture media containing fetal calf serum, prevented cell aggregation and neurite formation permanently (Figs 3, 4 and 5 left row), in a dose-dependent manner with the following potencies: spermine>spermidine>> putrescine (Fig. 6). PA had no effect on the adhesion of the cells to the polylysine coated surface as demonstrated at 4 and 16 hr after plating (Fig. 7). Recovery from the PA effects was observed after washing and changing to fresh culture medium not containing PA (Fig. 5 middle row).
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boys in culture Fig. 4, Changes in the numbers of neurons with neurites grown from the start in cultures containing fetal calf serum and in the presence of the drugs putrescine (Put, 3 raM), spermidine (Spd, I(X) i,tM), spermine (Spm. 50 IxM), and DFMO (5 mM), or in the absence of drugs (cofitrol). Neurons were considered to bear neurites when the extensions were equal to or longer than twice the cell body diameter. Results are expressed as percent of total cell number per dish. Each point represents the mean (-+ S.E.M.) wdue of 5 cultures in 3 separate experiments.
Effects of aminoguanidine A m i n o g u a n i d i n e c o u l d p r e v e n t the effects o f P A in a d o s e P A - d e p e n d e n t m a n n e r : p u t r e s c i n e < s p e r m i d i n e < s p e r m i n e (Fig, 5 right row). T h e p r e v e n t i v e effect o f a m i n o g u a n i d i n e c o u l d be d e m o n s t r a t e d b y its effect on t h y m i d i n e i n c o r p o r a t i o n by the c u l t u r e d cells. W h i l e P A d r a s t i c a l l y i n h i b i t e d t h y m i d i n e i n c o r p o r a t i o n , a m i n o g u a n i d i n e r e s t o r e d it within 2 d a y s (Fig. 8).
Effects of spermine on grown cultures A d d i t i o n o f 50 ixM s p e r m i n e to 24-hr-old o r o l d e r cultures, g r o w n in m e d i u m c o n t a i n i n g fetal calf s e r u m , h a d a c y t o l e t h a l effect (Fig. 9b). This lethal effect was d o s e - d e p e n d e n t a n d s t a r t e d to be a p p a r e n t b e t w e e n 5 a n d 10 txM (results not s h o w n ) . T h e c y t o l e t h a l effect c o u l d be p a r t i a l l y p r e v e n t e d by 5 m M a m i n o g u a n i d i n e (Fig. 9c).
Effects of PA in media containing sera from different species T h e a d v e r s e effects of P A on n e u r o n a l g r o w t h c o u l d be o b s e r v e d with m e d i a c o n t a i n i n g fetal calf s e r u m (Fig. 5 left r o w ) , h e a t i n a c t i v a t e d fetal calf s e r u m (Fig. 10 left row) a n d h u m a n s e r u m (Fig. 10 m i d d l e r o w ) , b u t not with rat s e r u m (Fig. 10 right row).
Polyamines and growth of cultured neurons
Fig. 2. Effects of D F M O (5 mM), present from plating time, on neuronal growth in 24 hr (c) and 48 hr (d) cultures containing fetal calf serum. Frames a and b are control cultures grown without DFMO for 24 and 48 hr, respectively. Original magnification = x 160. Bar = 120 p,M.
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Fig. 5. Left row: cultures grown for 24 hr: (a) in the absence of drugs (control); (b) with putrescine (3 mM); (c) with spermidine (100 p.M); and (d) with spermine (50 p.M). Middle row: cultures were grown for 24 hr in the presence or absence of drugs as above and then washed and grown for an additional 24 hr in fresh medium containing no drugs. (a) Control; (b) washed after putrescine; (c) washed after spermidine; and (d) washed after spermine. Right row: cultures grown for 24 hr with aminoguanidine (5 mM) (a) and with aminoguanidine in combination with putrescine (b), spermidine (c) and spermine (d). All drugs or drug combinations were added at plating time in the presence of fetal calf serum. Original magnification = × 160. Bar = 120 I~M.
P o l y a m i n e s a n d g r o w t h of c u l t u r e d n e u r o n s
Fig. 7. Effect of spermine (50 p,M), present from plating time, on dissociated cerebellar cells after 4 hr (b) and 16 hr (d) in cultures containing fetal calf serum. (a) and (c) Control cultures at 4 and 16 hr after plating, respectively. Note the beginning of neurite formation after 16 hr in controls (c). Original magnification --- x 160. Bar = 120 ~M.
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Fig. 9. Effect of spermine (50 ~.M) (b) and combination of spermine with aminoguanidine (5 mM) (c) on 24-hr-old cultures grown in the presence of fetal calf serum. Drugs were added 24 hr after plating time by changing the medium. Control cultures were grown for 48 hr in the absence of drugs (a). Original magnifications = (a) and (b) x 160; (c) x 250. Bars = 120 ~.M in (a) and (bL 75 ~M in (c).
P o l y a m i n e s and growth o f cultured n e u r o n s
Fig. 10. Effect of 3 mM putrescine (b), 100 ttM spermidine (c) and 50 ttM spermine (d) present from plating time in 24-hr-old cultures containing heat-inactivated fetal calf serum (left row), human serum (middle row) and rat serum (right row). Note the lack of PA cytostatic effects on neurons in the presence of rat serum as compared to controls (a). Original magnification = × 160. Bar = 120 ttM.
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Fig. 8. [3H]Thymidine incorporation into I- and 2-day-old cultures of dissociated cerebellar cells grown from plating time in media containing fetal calf serum, and 3 mM putrescine (Put), 100 ~M spermidine (Spd), 51) p,M spermine (Spin), combination of the above PA with 50 I,tM, 100 p,M and 5 mM aminoguanidine (Ag), respectively, 5 ~M Ara-C, or in the absence of drugs (control). Thymidine was added at plating time and after I day. Each point represents the mean ( - S.E.M.) value of 5 cultures in 3 separate experiments.
DISCUSSION Role o f PA in neuronal differentiation
The present study demonstrates that high ODC activity is present in cultured cerebellar neurons at the period of neurite outgrowth, indicating a demand for enhanced PA biosynthesis. Continuous irreversible inhibition of ODC from the start of cultures produced an initial delay in the growth of the cultured neuroblasts. It has been recently demonstrated by Seiler et al. 39 that DFMO, even at the high concentrations presently used (i.e. 5 mM) depletes the contents of putrescine and spermidine but not the concentrations of spermine. Furthermore, they observed no effects of DFMO on neuronal growth in primary cultures. 39 We were able to detect an early initial delay in the initiation of neuronal outgrowth in the presence of DFMO. The temporary effect of DFMO may be explained by its inability to deplete spermine 39 which perhaps can substitute for the other PA. The present findings therefore leave no doubt for an essential role of ODC activity in neuronal growth in culture. The exact cellular mechanisms of action of PA still remain elusive. They possibly may be involved in the regulation of selective protein syntheses or in the control of synthesis of other cellular components via stimulation or inhibition of specific enzymes. 10.24,42
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G.M. Gilad and V. H. Gilad
Effects of exogenous PA
A main purpose of the present study was to use the neuronal tissue culture technique as a model system to examine the effects of exogenous PA on survival and differentiation of neurons. The results demonstrate that exogenous PA exert reversible cytostatic effects on dissociated cerebellar neuroblasts upon culturing and irreversible cytolethal effects on grown neurons, in cultures containing fetal calf serum. The toxic effect of PA on cultured cells has been well known for quite some time 5"2°'22and is due to the presence of serum amine oxidase (copper dependent), the enzyme catalyzing the oxidative deamination of PA and the formation of toxic aminoaidehydes. 3.9.z3,33.43 This is corroborated by the present study demonstrating that: (1) the PA effect is not observed in the presence of rat serum, known to contain very low serum amine oxidase activity, 37 and (2) the diamine/polyamine oxidase inhibitor aminoguanidine can prevent the PA effect. The fact that heat inactivation of the fetal calf serum was not effective in preventing the toxicity may indicate that such a treatment is not sufficient to inactivate serum amine oxidase activity. The reason for the irreversible cytolethal effect of PA on grown cultured neurons with extended neurites, as opposed to the reversible cytostatic effects on neuroblasts, is not clear. One explanation may be the production of tissue polyamine oxidase (FAD-dependent) within these growing neurons, as has been demonstrated in cultured lymphocytes. 41 The fact that acetylation activity is known to be present in differentiated neurons, ~2 together with the demonstration that acetylpolyamines are probably better substrates for tissue polyamine oxidase, ~ lend further support to this explanation. Spermidine and spermine were much more potent, by about two orders of magnitude, than putrescine. This might be due to the formation of acrolein.2'27 This highly toxic aldehyde is formed by nonenzymatic [3-elimination of the unstable aminoaldehydes.~ Putrescine was toxic only in high concentrations, of above 2.5 mM. This could be the result of the formation of the toxic aldehyde and H202, catalyzed by the activity of diamine oxidase. 2¢~ Alternatively putrescine might have been taken up by the cultured neurons 25"34' and served as a precursor for the formation of spermidine and spermine, which could in turn be deaminated to form the toxic aldehydes. The fact that cadaverine, a substrate of diamine oxidase, 26'42 in concentrations above 2.5 raM, is also toxic (results not shown) lends support to the former explanation. Another mechanism of action suggested for the toxic effects of the aminoaldehydes, formed by the oxidative deamination of PA, is interference with D N A replication. 6 Cytosine arabinoside (Ara-c) inhibits D N A synthesis and thus prevents cell proliferation, but neurite formation is not affected by Ara-c. This indicates that although the polyamine degradative products do inhibit D N A replication as Ara-c does (Fig. 8), their primary mechanism of action in preventing neurite formation is on other cellular processes. In summary, the present study first demonstrates that O D C activity is essential for growth of cerebellar neuroblasts in culture, and second, indicates that oxidative deamination of PA and the formation of toxic products are the culprits in the cytotoxic effects of exogenous PA. The demonstration that exogenous PA are not toxic in the presence of rat serum and that the toxicity can be prevented with aminoguanidine, will serve the basis for further studies on the direct effects of PA on in vitro survival and growth of cultured neurons. These findings imply that the pharmacological effects of PA in v i v o can be expected to differ in different animal species. Acknowledgements--This research was supported by grants from the Muscular Dystrophy Association. the American
Paralysis Association and the Herman Goldman Foundation. Gad M. Gilad is an incumbent of the Paul and Gabriella Rosenbaum Career Development Chair in perpetuity, established by Paul and Gabriella Rosenbaum Foundation, Chicago, IL. REFERENCES 1. Alarcon R. A. (1971))Acrolein. IV. Evidence for the formation of the cytotoxicaldehyde acrolein from enzymatically oxidized spermine or spermidine. Archs Biochern. Biophys. 137, 365-372. 2. Alarcon R. A. (1972) Acrolein, a component of a universal cell-growth regulatory system: a theory. J. theor. Biol. 37, 159-167. 3. Allen J. C.. Smith C. J., Hussain J. 1., Thomas J. M. and Gaugas J. M. (1979) Inhibition of lymphocyteproliferation by polyamines requires ruminant plasma polyamine oxidase. Eur. J. Biochem. 107, 15,3-158. 4. Anderson T. R. and SchanbergS. M. (1972) Ornithine decarboxylase activity in developing rat brain. J. Nettrochem. 19, 1471-1476. 5. Bacbrach U. (1970) Oxidized polyamines. Ann. N. Y. Acad. Sci.. U.S.A. 1971, 939-948.
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6. Bachrach U. (19731 Function o f Nanlrally Occurring Polyamines. pp. 26-27. Academic Press, Inc., New York. 7. Black 1. B. and Mytilineou C. (19761 Transsynaptic regulation of the development of end organ innervation by sympathetic neurons. Brain Res. 101, 5(13-521. 8. Bolkenius F. N. and Seller N. (1981) Acetylderivatives as intermediates in polyamine catabolism. Int. J. Biochem. 13, 287-292. 9. Blaschko H. and Bonney R. (19621 Spermine oxidase and benzylamine oxidase. Distribution development and substrate specificity. Proc. R. Soc. Lond. BI56, 268-279. 10. Cohen S. S. (19711 hltroduction to the Polyamines. Prentice-Hall Inc., Englewood Cliffs, New Jersey. I I. Gahl W. A. and Pitot H. C. (1982) Polyamine degradation in fetal and adult bovine serum. Biochem. J. 202, 603-611. 12. Gaugas M. J., ed. (19801 Polyamines in Biomedical Research. Wiley, New York. 13. Gilad G. M. and Gilad V. H. (19811 Cytochemical localization of ornithine decarboxylase with rhodamine or biotinlabeled a-difluoromethylornithine. An example for the use of labeled irreversible enzyme inhibitors as cytochemical markers. J. Histochem. Cytochem. 29, 687-692. 14. Gilad G. M. and Gilad V. H. (19831 Visualization of rhodamine-labeled ct-difluoromethylornithine in viable neuroblasts: towards in vivo localization of ornithine decarboxylase. In Advances in Polyamine Research, Vol. 4 (eds Bachrach U., Kay A. and Chayen R.), pp. 585-590. Raven Press, New York. 15. Gilad G. M. and Gilad V. H. (1983) Early rapid and transient increase in ornithine decarboxylase activity within sympathetic neurons after axonal injury. Exp. Neurol. Ill, 158-166. 16. Gilad G. M. and Kopin I. J. (1979) Neurochemical aspects of neuronal ontogenesis in the developing rat cerebellum: changes in neurotransmitter and polyamine synthesizing enzymes. J. Neurochem. 33, 1195-1204. 17. Gilad G. M.. Carstairs J. and Gilad V. H. (19831 Biochemical compensation and recovery following temporary inhibition of ornithine decarboxylase during the development of rat cerebellar cortex. Int. J. Devl Neurosci. I, 297-304. t8. Gilad G. M., Dornay M. and Gilad V. H. 0985) Polyamine treatment in early development leads to increased numbers of rat sympathetic neurons. Brain Res., 348, 363-366. 19. Green L. A. and McGuire J. C. (19781 Induction of ornithine decarboxylase by nerve growth factor dissociated from effects on survival and neurite outgrowth. Nature, Lond. 276, 191-194. 20. Halevy S., Fuchs Z. and Mager J. (1962) Toxicity of spermine and spermidine for tissue cultures. Proc. Israel Chem. Soc. Bull,, Research Council o f Israel I IA, 52-53. 21. Hendry I. A. (1977) Cell division in the developing sympathetic nervous system. J. Neurocytol. 6, 299-3(19. 22. Higgins M. L., Tillman M. C., Rupp J. P. and Leach F. R. (19691 The effect of polyamines on cell culture cells. J. Cell Physiol. 74, 149-154. 23. Hirsch J. G. (19531 Spermine oxidase: an amine oxidase with specificity for spermine and spermidine. J. exp. Med. 97, 345-355. 24. J/inne J., P6s6 H. and Raina A. (1978) Polyamines in rapid growth and cancer. Biochim. biophys. Acta 473, 241-293. 25. J/,inne J., AIhonen-Honigsto L., Seppanen P. and H61t[i E. (1981) Cellular compensatory mechanisms during chemically induced polyamine deprivation. In Advances on Polyamine Research, Vol. 3 (eds Calderera C. M., Zappia V. and Bachrach U.), pp. 85-95. Raven Press, New York. 26. Kapeller-Adler R, (1970) Amine Oxidases and Methods for their Study. Wiley lnterscience, New York. 27. Kimes B. W. and Morris D. R. (19711 Inhibition of nucleic acid and protein synthesis in Escherichia coil by oxidized polyamines and acrolein. Biochim. biophys. Acta 228, 235-244. 28. Kingsbury A. E., Gallo V., Woodhams P. L. and Balazs R. (1985) Survival, morphology and adhesion properties of cerebellar interneurons cultured in chemically defined and serum-supplemented medium. Devl Brain Res. 17, 17-25. 29. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. 30. Messer A. and Smith D. (1977) hi vitro behavior of granule cells from granular neurological mutants of mice. Brain Res. 130, 13-23. 31. Metcalf B. W., Bey P., Danzin C., Jung M. J., Casara P. and Vevert J. P. (19781 Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E.C. 4. I. I. 17) by substrate and product analogs. J. Am. Chem. Soc. 100, 25512553. 32. Ortiz J. G., Giacobini E. and Schmidt- Glenewinkel T. (1983) Acetylation of polyamines in mouse brain: subcellular and regional distribution. J. Neurosci. Res. 9, 193-2111. 33. Otsuka H. (1971) The toxic effect of spermidine on normal and transformed cells. J. Cell Sci. 9, 71-84. 34. Pohjanpelto P. (19761 Putrescine transport is greatly increased in human fibroblasts initiate to proliferate. J. Cell Biol. 68, 512-5211. 35. Russell D. H., ed. (19731 Polyamines in Normal and Neoplastic Growth, Raven Press, New York. 36. Seller N. and Lamberty W. (19751 Interrelation between polyamines and nucleic acids: changes of polyamines and nucleic acid concentrations in the developing rat brain. J. Neurochem. 24, 5-13. 37. Seller N., Bolkenius F. N., Kn6dgen B. and Mamont P. (1980) Polyamine oxidase in rat tissues. Biochhn. biophys. Acta 615, 480--488. 38. Seller N., KnOdgen B., Bink G., Sarhan S. and Bolkenius F. (1983) Diamine oxidase and polyamine catabolism. In Advances in Polyamhw Research, Vol. 4 (eds Bachrach U., Kaye A. and Chayen R.), pp. 135-154. Raven Press, New York 39. Seller N., Sarhan S. and Roth-Schechter B. F. (1984) Polyamines and the development of isolated neurons in cell culture. Neurochem. Res. 9, 871-885. 40. Shore P. A. and Cohn V. H., Jr (19601 Comparative effects of monoamine oxidase inhibitors on monoamine oxidase and diamine oxidase. Biochem. Pharmac. 5, 91-95. 41. Smith C. J., Maschler R., Maurer H. R. and Allen J. C. (19831 Inhibition of cells in culture by polyamines does not depend on the presence of ruminant serum. Cell Tiss. Kinet. 16, 269--276. 42. Tabor H. and Tabor C. W. (19721 Biosynthesis and metabolism of 1,4-diaminobutane, spermidine, spermine and 3 related amines. Adv. Enzymol. 36, 203-268. 43. Tabor C. W., Tabor H. and Bachrach U. (19641 Identification of the aminoaldehydes produced by the oxidation of spermine and spermidine in purified plasma amine oxidase. J. biol. Chem. 239, 2194-2203. 44. Trenkner E. and Sidman R. L. ( 19771 Histogenesis of mouse cerebellum in microwell cultures: cell reaggregation and migration, fiber and synapse formation. J. Cell Biol. 75, 915-925.
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45. Wallace H. M. and Keir H. M. (1981) Uptake and excretion of polyamines from baby hamster kidney cells (BHK-21/ C13) the effect of serum on confluent cell cultures. Biochim. biophys. Acta 676, 25-30. 46. Yavin Z. and Yavin E. (1980) Survival and maturation of cerebral neurons on poly 0~-Iysine) surfaces in the absence of serum. Devl Biol. 75, 454-459.
Int. J. Devl. Neuroscience. Vol. 4, No. 3, pp. 195-2¢18,1986.
Printed in Great Britain.
POLYAMINES AFFECT GROWTH OF C U L T U R E D R A T C E R E B E L L A R N E U R O N S IN DIFFERENT SERA GAD M. GILAD and VARDA H. GILAD The Center for Neuroscience and Behavioral Research and The Department of Isotope Research, The Weizmann Institute of Science, 76100 Rehovot, Israel (Accepted 4 December 1985) A b s t r a c t - - T h e study examines the effects of polyamines on growth of cultured neurons from 6-day-old
rat cerebellar cortex, by means of: (a) irreversible inhibition of ornithine decarboxylase activity with a-difluoromethylornithine, and (b) treatment with the exogenous diamine putrescine and the polyamines spermidine and spermine, in the presence of sera from different sources. Inhibition of ornithine decarboxylase activity starting at plating time led after 24 hr to a partial inhibition of cell aggregation with a drastic (90%) inhibition of neurite formation. However, after 48 hr of enzyme inhibition aggregation and neurite formation increased to approach the 24 hr control values and eventually cultures fully recovered. Polyamines added at plating time in the presence of fetal calf serum led to a permanent dose-dependent inhibition of aggregation and neurite formation, spermine being effective at lower doses (spermine < spermidine ,~ putrescine). Adhesion of cells to polylysine coated surfaces was not affected by polyamines. Recovery from the cytostatic polyamine effect was observed after washing and addition of fresh medium. Prevention of the effect was achieved in the presence of aminoguanidine, an inhibitor of diamine and polyamine oxidases. The preventive effect of aminoguanidine was dose polyamine-dependent, with higher aminoguanidine concentrations needed to prevent the spermine effect (spermine->spermidine > putrescine). The polyamine effects were observed in the presence of fetal calf, heat-inactivated fetal calf and human sera, but not with rat serum. Addition of polyamines to 24-hr-old cultured neurons, in the presence of fetal calf serum, led 12 hr later to cell death. This lethal effect could be inhibited by aminoguanidine. We conclude: (a) irreversible inhibition of ornithine decarboxylase activity delays but does not prevent neuronal growth in culture; (b) oxidation products of extracellular polyamines inhibit cell aggregation and neurite formation of cultured neuroblasts, and have lethal effects on growing neurons in culture, and (c) different pharmacological effects of polyamines can be expected in different species. Key words: Polyamines, Cerebellar cultures, Sera.
T h e b i o g e n i c p o l y a m i n e s ( P A ) s p e r m i d i n e a n d s p e r m i n e , a n d their d i a m i n e p r e c u r s o r p u t r e s c i n e , have b e e n s h o w n to be p r e r e q u i s i t e s for o p t i m a l cellular g r o w t h . 1°'12"24"35"42 P o l y a m i n e s were d e m o n s t r a t e d to affect v a r i o u s r e g u l a t o r y m o l e c u l a r m e c h a n i s m s i n c l u d i n g those of D N A , R N A a n d p r o t e i n synthesis. 1°'24"42 In the b r a i n , P A b i o s y n t h e s i s has b e e n s h o w n to increase rapidly prior to o n s e t of growth. 4"36 It has b e e n r e c e n t l y d e m o n s t r a t e d that selective i n h i b i t i o n of o r n i t h i n e d e c a r b o x y l a s e ( O D C ) activity, the e n z y m e catalyzing the first step in P A biosynthesis, with p u t r e s c i n e a n a l o g s causes i n h i b i t i o n of n e u r i t e f o r m a t i o n by c u l t u r e d chick n e u r o b l a s t s . 39 This growth i n h i b i t i o n can be r e v e r s e d by p u t r e s c i n e i n d i c a t i n g i m p o r t a n t role for P A in n e u r o n a l d i f f e r e n t i a t i o n . This r e c e n t s t u d y indicates that the growth characteristics of n o r m a l n e u r o n s m a y differ from the growth of n e o p l a s t i c p h a e o c h r o m o c y t o m a cells w h e r e i n h i b i t i o n of O D C activity with an a n a l o g of o r n i t h i n e , did n o t affect n e u r i t e outgrowth.19 Paradoxically, h o w e v e r , while i n t r a c e l l u l a r P A are essential for o p t i m a l cell growth, e x o g e n o u s P A can e v o k e p o t e n t s u p p r e s s i o n of cell p r o l i f e r a t i o n a n d cytotoxicity w h e n a d d e d to cell cultures in the p r e s e n c e of r u m i n a n t sera. 5"2°'22 This adverse effect of P A is m a i n l y d u e to the oxidative d e a m i n a t i o n of P A a n d the f o r m a t i o n of a m i n o a l d e h y d e s by p o l y a m i n e oxidase p r e s e n t in these sera.3,9,23,33.43 W e have r e c e n t l y d e m o n s t r a t e d that O D C activity is e l e v a t e d in rat s y m p a t h e t i c n e u r o n s d u r i n g the p e r i n a t a l p e r i o d 15 w h e n n e u r o n a l cell division e n d s a n d n a t u r a l l y o c c u r r i n g n e u r o n a l cell d e a t h b e g i n s ; 21 a p e r i o d which coincides with late stages of growth of t e r m i n a l axons within target tissues. 7 T h e s e findings have suggested that there is an increased d e m a n d for P A d u r i n g that p e r i o d a n d this is reflected by e n h a n c e d P A biosynthesis. B a s e d o n this r a t i o n a l e we have Abbreviations: Ag, aminoguanidine; Ara-c, cytosine arabinoside; DFMO, ct-difluoromethylornithine; ODC, ornithine deearboxylase; PA. polyamines; Put, putrescine, Spd, spermidine; Spin, spermine. DN 4:3-A
195
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G . M . Gilad and V. H. Gilad
treated rats with exogenous PA for a limited postnatal period and demonstrated that it can lead to an increased number of sympathetic neurons.l~ It is unknown whether PA exert their in vivo effect directly on the neurons themselves, or indirectly through interaction with other cells which in turn exert beneficial effect on the developing neurons. Tissue culture studies may clarify some of these questions. Indeed, the relative ease with which the development of rat cerebellar neuroblasts in cultures can be studied, 28"3°'44together with the indication of the possible involvement of PA in the differentiation of cerebellar interneurons, j4"~617 make such cultures an attractive experimental model for studies of PA pharmacology. In the present study therefore we examined the role of PA in growth of cultured cerebellar neurons using two approaches: (1) inhibition of PA biosynthesis in cultured neurons with e~-difluoromethylornithine ( D F M O ) , an enzyme-activated irreversible inhibitor of O D E 31 and (2) cultivation of neurons with media containing exogenous PA in the presence of: (a) aminoguanidine, an inhibitor of diamine oxidase 4° and, at higher concentrations, of polyamine oxidase,ll'38 and (b) sera from different species. EXPERIMENTAL PROCEDURES
Materials Putrescine dihydrochloride, spermidine trihydrochloride, spermine tetrahydrochloride, aminoguanidine bicarbonate, cytosine arabinoside hydrochloride (Ara-C) and poly-L-iysine hydrobromide (mol. wt 30,000-70,000) were all from Sigma Chemical Co., St. Louis, MO, U.S.A. Ornithine DL-[I-t4C] (sp. act. 49 mCi/mmol) and thymidine [methyl-3H] (sp. act. 6.7 Ci/ mol) were from New England Nuclear, Dreieich, West Germany. Dulbecco's modified Eagle medium and fetal calf serum were from biological Industries Beth H a E m e k , Israel; miramycin (antibiotic) was from Teva, Jerusalem, Israel. DL-et-Difluoromethylornithine ( M D L 71782), was a generous gift from Centre de Recherche Merell International subsidiary of the Dow Chemical Company, Strasbourg, France. Sera Rat trunk blood was collected after decapitation. Human blood was collected by venipuncture. Blood samples were allowed to clot and the sera collected and sterilized by membrane filtration. Sera were heat-inactivated at 57°C for 30 min. Cell culture The brain was rapidly removed from 6-day-old rat pups after decapitation and the cerebellum dissected ~7 and transferred aseptically to a 15 ml sterile plastic conical tube containing 3 ml of Dulbecco's modified Eagle medium containing 10% serum, 2 mM glutamine and 0.1% miramycin. Six to 8 cerebella were pooled together and prepared for culture by adapting the method of Yavin and Yavin. 46 The cerebellum was mechanically dissociated after 15-20 passages through a sterile 13 Ga. 10 cm long stainless steel needle connected through a nylon sieve (48 Ixm pore size) to a 5 ml sterile syringe. The dissociated cerebellum was allowed to settle for a few minutes and the supernatant containing the dissociated cells was collected and centrifuged at 200 g for 5 min. The pellet was resuspended in fresh medium, the cells were counted in a haemocytometer and their viability determined by the Trypan Blue exclusion test. The dissociated cells were cultured (400/m 2) on polylysine coated 46 plastic Petri dishes, or glass coverslips situated in 35 mm plastic Petri dishes. The cultures were incubated at 37°C in an atmosphere of 5% CO2 in air and 100% humidity. Drugs or drug combinations were either added from the beginning, or 24 hr after the beginning of cultures. Cultures were regularly examined in an inverted microscope and photographed with phase contrast microscopy. Thymidine incorporation Medium containing [3H]thymidine, 0.6 I~Ci/dish, was added at plating time or 24 hr after plating. Twenty four hours after thymidine addition cultures were washed twice with fresh
Polyamines and growth of cultured neurons
197
medium, the cells scraped off, transferred to counting vials and radioactivity was measured by scintillation spectrometry.
Biochemical assays Cells were scraped off the culture dish and homogenized in 100 mM Tris-HCI buffer (pH 7.4) containing 7.5 mM dithiothreitol, 6 mM ethylenediamine tetraacetic acid (EDTA) and 40 IxM pyridoxal-5'-phosphate. ODC activity was assayed as previously described.'3 Protein concentrations were determined according to the method of Lowry et ai.29 RESULTS
Effects of DFMO ODC activity was lower at the beginning of culture than in age matched cerebellar homogenates (Fig. 1). However, the enzyme activity was transiently elevated with a peak at 24 hr. The activity was still high at 2 days when about 93% of neurons had already extended neurites (Fig. 4), and when thymidine incorporation was low (Fig. 8). By 3 days ODC activity declined to very low levels (Fig. 1).
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Fig. 1. Time-course of changes in ODC activity in cultures of cerebellar cells containing fetal calf serum in the absence (control, solid circle) or in the presence of 5 mM DFMO (open circle). The activity in the homogenate of 6-day-old cerebellum is presented (original homogenate, solid circle). Each point represents the mean ( - S . E . M . ) value of 3 separate experiments with 5 separate determinations (culture dishes) each.
The continuous presence of 5 mM DFMO, from the start, in culture media containing fetal calf serum, completely inhibited ODC activity for the 3 days duration of the experiment (Fig. 1). Morphologically, DFMO led to about 24 hr delay in cell aggregation and neurite formation (Figs 2-4).
Effects of early addition PA The continuous presence of putrescine or PA, from the start, in culture media containing fetal calf serum, prevented cell aggregation and neurite formation permanently (Figs 3, 4 and 5 left row), in a dose-dependent manner with the following potencies: spermine>spermidine>> putrescine (Fig. 6). PA had no effect on the adhesion of the cells to the polylysine coated surface as demonstrated at 4 and 16 hr after plating (Fig. 7). Recovery from the PA effects was observed after washing and changing to fresh culture medium not containing PA (Fig. 5 middle row).
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Effects of aminoguanidine A m i n o g u a n i d i n e c o u l d p r e v e n t the effects o f P A in a d o s e P A - d e p e n d e n t m a n n e r : p u t r e s c i n e < s p e r m i d i n e < s p e r m i n e (Fig, 5 right row). T h e p r e v e n t i v e effect o f a m i n o g u a n i d i n e c o u l d be d e m o n s t r a t e d b y its effect on t h y m i d i n e i n c o r p o r a t i o n by the c u l t u r e d cells. W h i l e P A d r a s t i c a l l y i n h i b i t e d t h y m i d i n e i n c o r p o r a t i o n , a m i n o g u a n i d i n e r e s t o r e d it within 2 d a y s (Fig. 8).
Effects of spermine on grown cultures A d d i t i o n o f 50 ixM s p e r m i n e to 24-hr-old o r o l d e r cultures, g r o w n in m e d i u m c o n t a i n i n g fetal calf s e r u m , h a d a c y t o l e t h a l effect (Fig. 9b). This lethal effect was d o s e - d e p e n d e n t a n d s t a r t e d to be a p p a r e n t b e t w e e n 5 a n d 10 txM (results not s h o w n ) . T h e c y t o l e t h a l effect c o u l d be p a r t i a l l y p r e v e n t e d by 5 m M a m i n o g u a n i d i n e (Fig. 9c).
Effects of PA in media containing sera from different species T h e a d v e r s e effects of P A on n e u r o n a l g r o w t h c o u l d be o b s e r v e d with m e d i a c o n t a i n i n g fetal calf s e r u m (Fig. 5 left r o w ) , h e a t i n a c t i v a t e d fetal calf s e r u m (Fig. 10 left row) a n d h u m a n s e r u m (Fig. 10 m i d d l e r o w ) , b u t not with rat s e r u m (Fig. 10 right row).
Polyamines and growth of cultured neurons
Fig. 2. Effects of D F M O (5 mM), present from plating time, on neuronal growth in 24 hr (c) and 48 hr (d) cultures containing fetal calf serum. Frames a and b are control cultures grown without DFMO for 24 and 48 hr, respectively. Original magnification = x 160. Bar = 120 p,M.
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Fig. 5. Left row: cultures grown for 24 hr: (a) in the absence of drugs (control); (b) with putrescine (3 mM); (c) with spermidine (100 p.M); and (d) with spermine (50 p.M). Middle row: cultures were grown for 24 hr in the presence or absence of drugs as above and then washed and grown for an additional 24 hr in fresh medium containing no drugs. (a) Control; (b) washed after putrescine; (c) washed after spermidine; and (d) washed after spermine. Right row: cultures grown for 24 hr with aminoguanidine (5 mM) (a) and with aminoguanidine in combination with putrescine (b), spermidine (c) and spermine (d). All drugs or drug combinations were added at plating time in the presence of fetal calf serum. Original magnification = × 160. Bar = 120 I~M.
P o l y a m i n e s a n d g r o w t h of c u l t u r e d n e u r o n s
Fig. 7. Effect of spermine (50 p,M), present from plating time, on dissociated cerebellar cells after 4 hr (b) and 16 hr (d) in cultures containing fetal calf serum. (a) and (c) Control cultures at 4 and 16 hr after plating, respectively. Note the beginning of neurite formation after 16 hr in controls (c). Original magnification --- x 160. Bar = 120 ~M.
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Fig. 9. Effect of spermine (50 ~.M) (b) and combination of spermine with aminoguanidine (5 mM) (c) on 24-hr-old cultures grown in the presence of fetal calf serum. Drugs were added 24 hr after plating time by changing the medium. Control cultures were grown for 48 hr in the absence of drugs (a). Original magnifications = (a) and (b) x 160; (c) x 250. Bars = 120 ~.M in (a) and (bL 75 ~M in (c).
P o l y a m i n e s and growth o f cultured n e u r o n s
Fig. 10. Effect of 3 mM putrescine (b), 100 ttM spermidine (c) and 50 ttM spermine (d) present from plating time in 24-hr-old cultures containing heat-inactivated fetal calf serum (left row), human serum (middle row) and rat serum (right row). Note the lack of PA cytostatic effects on neurons in the presence of rat serum as compared to controls (a). Original magnification = × 160. Bar = 120 ttM.
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DISCUSSION Role o f PA in neuronal differentiation
The present study demonstrates that high ODC activity is present in cultured cerebellar neurons at the period of neurite outgrowth, indicating a demand for enhanced PA biosynthesis. Continuous irreversible inhibition of ODC from the start of cultures produced an initial delay in the growth of the cultured neuroblasts. It has been recently demonstrated by Seiler et al. 39 that DFMO, even at the high concentrations presently used (i.e. 5 mM) depletes the contents of putrescine and spermidine but not the concentrations of spermine. Furthermore, they observed no effects of DFMO on neuronal growth in primary cultures. 39 We were able to detect an early initial delay in the initiation of neuronal outgrowth in the presence of DFMO. The temporary effect of DFMO may be explained by its inability to deplete spermine 39 which perhaps can substitute for the other PA. The present findings therefore leave no doubt for an essential role of ODC activity in neuronal growth in culture. The exact cellular mechanisms of action of PA still remain elusive. They possibly may be involved in the regulation of selective protein syntheses or in the control of synthesis of other cellular components via stimulation or inhibition of specific enzymes. 10.24,42
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G.M. Gilad and V. H. Gilad
Effects of exogenous PA
A main purpose of the present study was to use the neuronal tissue culture technique as a model system to examine the effects of exogenous PA on survival and differentiation of neurons. The results demonstrate that exogenous PA exert reversible cytostatic effects on dissociated cerebellar neuroblasts upon culturing and irreversible cytolethal effects on grown neurons, in cultures containing fetal calf serum. The toxic effect of PA on cultured cells has been well known for quite some time 5"2°'22and is due to the presence of serum amine oxidase (copper dependent), the enzyme catalyzing the oxidative deamination of PA and the formation of toxic aminoaidehydes. 3.9.z3,33.43 This is corroborated by the present study demonstrating that: (1) the PA effect is not observed in the presence of rat serum, known to contain very low serum amine oxidase activity, 37 and (2) the diamine/polyamine oxidase inhibitor aminoguanidine can prevent the PA effect. The fact that heat inactivation of the fetal calf serum was not effective in preventing the toxicity may indicate that such a treatment is not sufficient to inactivate serum amine oxidase activity. The reason for the irreversible cytolethal effect of PA on grown cultured neurons with extended neurites, as opposed to the reversible cytostatic effects on neuroblasts, is not clear. One explanation may be the production of tissue polyamine oxidase (FAD-dependent) within these growing neurons, as has been demonstrated in cultured lymphocytes. 41 The fact that acetylation activity is known to be present in differentiated neurons, ~2 together with the demonstration that acetylpolyamines are probably better substrates for tissue polyamine oxidase, ~ lend further support to this explanation. Spermidine and spermine were much more potent, by about two orders of magnitude, than putrescine. This might be due to the formation of acrolein.2'27 This highly toxic aldehyde is formed by nonenzymatic [3-elimination of the unstable aminoaldehydes.~ Putrescine was toxic only in high concentrations, of above 2.5 mM. This could be the result of the formation of the toxic aldehyde and H202, catalyzed by the activity of diamine oxidase. 2¢~ Alternatively putrescine might have been taken up by the cultured neurons 25"34' and served as a precursor for the formation of spermidine and spermine, which could in turn be deaminated to form the toxic aldehydes. The fact that cadaverine, a substrate of diamine oxidase, 26'42 in concentrations above 2.5 raM, is also toxic (results not shown) lends support to the former explanation. Another mechanism of action suggested for the toxic effects of the aminoaldehydes, formed by the oxidative deamination of PA, is interference with D N A replication. 6 Cytosine arabinoside (Ara-c) inhibits D N A synthesis and thus prevents cell proliferation, but neurite formation is not affected by Ara-c. This indicates that although the polyamine degradative products do inhibit D N A replication as Ara-c does (Fig. 8), their primary mechanism of action in preventing neurite formation is on other cellular processes. In summary, the present study first demonstrates that O D C activity is essential for growth of cerebellar neuroblasts in culture, and second, indicates that oxidative deamination of PA and the formation of toxic products are the culprits in the cytotoxic effects of exogenous PA. The demonstration that exogenous PA are not toxic in the presence of rat serum and that the toxicity can be prevented with aminoguanidine, will serve the basis for further studies on the direct effects of PA on in vitro survival and growth of cultured neurons. These findings imply that the pharmacological effects of PA in v i v o can be expected to differ in different animal species. Acknowledgements--This research was supported by grants from the Muscular Dystrophy Association. the American
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