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Cytotoxic effects of monodansylcadaverine and methylamine in primary cultures of rat cerebellar neurons
SHORT COMMUNICATION CYTOTOXIC EFFECTS OF MONODANSYLCADAVERINE AND METHYLAMINE IN PRIMARY CULTURES OF RAT CEREBELLAR NEURONS GAD The Center
for Neurosciences
M.
GILAD*
and Behavioral
and
Research.
VARDA
H.
GILAD*
The Weizmann
Institute
of Science.
Rehovot.
Israel
( Accqwd 22 ~ffrciz 19%) Abstract-The effects of dansylcadaverine and methylamine. competitive inhibitors of transglutaminase. were examined in primary cultures of dissociated rat ccrehcllar neurons. Addition of the drugs at pfating time resulted 24 hr later in irreversible cytotoxic effects evidenced by failure of aggregation and neurite formation. Cytotoxicity was dose-dependent with methylamine being more potent (I(‘~,,= 20 PM) than dansylcadaverine (I(‘ 5,, = 30 PM). The cytotoxic effects were less potent when drugs were added 24 hr after plating. the time when neurons had already begun to extend neurites. Drugs were effective in the various sera and heat-inactivated sera tested. We concluded that low doses of methylamine and dansylcadaverine have potent toxic effects on primary neuronal cultures. Cerehellar K(~J H*oT(/.s: Transglutaminase inhibitors. Methylamine. Cerebellum. Transglutaminasc.
cultures.
Cytotoxicity,
Monodansylcadaverine.
Monodansylcadaverine (DC) and methylamine (MA) are well-known competitive inhibitors of the enzyme transglutaminase (TG)‘.“‘. which catalyzes the covalent binding of small molecular have been weight primary amines to glutamine residues on polypeptides. ‘.I3 These inhibitors used in recent studies as indicators for TG participation in various biological functions.‘.‘.7-X Membrane associated TG activity was implicated in several developmental functions,* such as receptor-mediated endocytosisX and cell-substrate interactions (adhesion).‘.’ High TG activity was recently observed during early stages of neuronal growth in the rat cerebellum.’ In the present study therefore we sought to characterize the effects of DC and MA on dissociated rat cerebellar neurons in primary cultures, where neuronal growth and differentiation can be closely followed under controlled conditions.“-‘“.” Primary cultures were prepared from fi-day-old mechanically dissociated rat cerebellum, as reported before.” The dissociated cells were plated (400/mm2) on polylysine-coated glass coverslips” situated in 35 mm plastic Petri dishes. Drugs or drug combinations were added at plating time, or 24 hr after plating. DC and MA were purchased from Sigma Chemical Co., St Louis, MO, U.S.A. DC was dissolved in minimal volume of dimethyl sulfoxide (DMSO) and diluted to the final concentration with the culture medium. At the lowest dilution, DMSO alone (0.5% v/v) had no effect on cultures. For biochemical assays ceils were scraped off the culture dish and homogenized in 50 mM Tris-HCI buffer (pH 7.4) containing 7.5 mM dithiothreitol. TG activity was assayed as previously described.” Protein concentrations were determined according to the method of Lowry ef al.” The continuous presence of DC or MA, from plating time, prevented cell aggregation, neurite formation and caused cell death (Fig. 1, left and middle rows) in a dose-dependent manner with MA being more potent (IC~Q= 20 PM) than DC (ICY,,= 33 wM) (Fig. 2). Cytolethal effects of MA (50 PM) were observed as early as 4 hr after plating (Fig. 1, left row). No early effects of DC (50 PM) were observed, probably indicating that DC did not affect the attachment of cells to the polylysine-coated surface. The cytotoxic effects of DC and MA were not dependent on the serum used. Similar effects were observed in media containing fetal calf, rat, or human sera, which were either normal or heat-inactivated (results not shown). A~br~~iurj~~i.s: DC, monodansyi~daverine~ Prchcnt address: Veterans Administration Wars Parkway. Boston. MA 02132. U.S.A.
DN 4:5-A
MA. methylamjne; TG, transglut~lmin~l5c. Medical Center. Spinal Cord Injury Research.
401
1400 Veterans
of Foreign
402
G. M.
Glad
and
IO Amine
V. H.
Gilad
too concentration
1000
(p M)
Fig. 2. Dose-dependent effect of MA and DC on the numbers of neurcms bearing neurrtes in 24hr-old cultures. Drugs were present from plating time. Neurons were considered to hear neurites when the extensions were equal to or longer than twice the cell body size. Results are expressed as percent of total cell number per dish. Each point represents the mean value of 3 cultures in 2 separate experiments (SD. values did not exceed 20% of mean values).
The cytotoxic effects of the drugs were irreversible, as no recovery was detected after washing and changing to fresh medium not containing drugs (Fig. 1. right row). The present study demonstrates that DC and MA exert irreversible cytotoxic effects on dissociated cultured rat cerebellar neuroblasts, which are evidenced by failure of cell aggregation and neurite formation. eventually leading to cell death. MA is more potent and its effects occur earlier than DC. The effects of these drugs are less potent when added to 24- or 48-hr-old cultures containing neurons with extended neurites (Fig. 3). Previous studies with these TG inhibitors have indicated that membrane associated TG activity may participate in several developmental functions,’ such as receptor-mediated endocytosis” and cell-substratum interactions (adhesion). ‘.’ It is noteworthy, however, that the dose-dependent cytotoxic effects presently observed to occur after many hours, can be achieved with much lower concentrations than those needed to inhibit short-term processes, such as receptor-mediated endocytosis,’ where TG activity has been implicated. In the brain a substantial amount of TG activity may be associated with or even present on neuronal membranes.” TG activity in our cerebellar cultures was about 100 pmol/mg protein/hr at plating time and nearly doubled in 24”hr-old or older cultures. The demonstration that cell aggregation and neurite formation are inhibited by DC and MA. two processes which require cell-cell and cell-substratum interactions, imply that membrane associated TG activity may play a role in these processes. Yet, the adverse drug effects on growth might have been secondary to cell death, therefore the effects of sublethal doses of DC and MA on TG activity in cultures will have to be determined. Polylysine, used in this study to coat the growth surfaces, is a poor substrate for TG (G. M. Gilad and L. E. Varon, unpublished observations), but other extracellular components, such as fibronectin, a known substrate of TG,lJ may serve as substrates in such interactions. Interestingly, substantial TG activity is present in the various sera tested. Solutions containing 10% sera had average activity of 22.5 2 1.5 pmollmg protein/hr. Further, heat inactivation led only to a reduction of about 32% in serum TG activity. This finding raises the possibility that serum TG activity may participate in some aspects of cell development at least in tissue culture. Polyamines, known substrates of TG.“.“’ are present in the brain in high concentrations during and inhibition of polyamine biosynthesis in dissociated embryonic CNS cultures development,” can inhibit neuronal development.5%‘” These findings indicate that DC and MA may exert some of their effects by competing with the natural polyamines as substrates for TG catalyzed reactions. it would be interesting therefore to determine whether or not polyamines can prevent the cytotoxic effects of the drugs.
Dansylcadaverine
and methylamine
in cerebellar
cultures
403
Fig. 3. Effects of SO WM MA (h) and SO FM DC (c) on 2.bhr-old cultures. Drugs were added 24 hr after plating hy changing the medium and cultures were grown for additional 24 hr. Controls (a) were grown for 4X hr in the ahsenoc of drugs. Orlgin:ll mngnilication = x IhO. Bar = 120 FM.
Dansylcadaverine
and methylamine
in cerebellar cultures
405
In summary, MA and DC have potent toxic effects on primary cultures of rat cerebellar neurons. The primary cellular effects of these drugs are unknown. Calcium activated TG is one process which has been implicated, due to the competitive inhibition exerted on TG activity by MA and DC. Acknow/~r/~cmc,rr/s-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 Bersten A. M.. Ahkong 0. F.. Hallinan T.. Nelson S. J. and Lucy J. A. (19X3) inhibition of the formation of myotubes irr virro by inhibitors of transglutaminase. Biorhim. hiophys. Acfcr 762, 429-436. catalyzed incorporation of putrescine into surface proteins of mouse neuro2. Chen Y. K. (1984) Transglutaminase blastoma cells. Molec. cell. Biochem. 58, Yl-97. 3. Comwell M. M.. Juliano R. L. and Davies P. J. A. (1983) inhibition of the adhesion of Chinese hamsterovary cells by the naphytylsulfonamides dansylcadaverine and N-(6-aminohexyl)-5-chloro-l-naphthylenesulf~~namide (~7). Biochim. hiophys. Actor 762, 414-Il9. Anu. Ret,. Biochon. 49, 517-531. 4. Folk J. F. (19X0) Transglutaminases. 5. Gilad G. M. and Gilad V. H. (1986) Polyamines affect growth of cultured rat cerebellar neurons in different sera. Inr. J. Devl Neurosci. 4, 195-208. activity in rat brain: characterization. distribution and changes 6. Gilad G. M. and Varon L. E. (1985) Transglutaminase with age. J. Nerrrorhertr. 45, 1522-1526. V.. Schapf R. E., Hanauske-Abel H. M. and Schulte-Wissermann H. (1982) Transglutaminase and 7. Gunzler polyamine dependence of effector functions of human immunocompetent cells. The effect of specific inhibitors on lymphocyte proliferation and granulocyte chemiluminescence. FEBS Let/. 150, 3Y0-396. inhibits internalization of 8. Haigler H. T.. Maxfield F. R.. Willingham M. C. and Pastan I. (1979) Dansylcadaverine ‘“SI-epidermal growth factor in BALB 3T3 cells. J. hiol. Chem. 255, 1239-1241. 9. Kingsbury A. E.. Gallo V., Woodhams P. L. and Balazs R. (IYgS) Survival. morphology and adhesion properties of cerebellar interneurons cultured in chemically defined and serum-supplemented medium. Devl Brnirl Res. 17, 17-2.5. Mokec. cc/l. Biochem. 58, Y-3.5. IO. Lorand L. and Conrad S. M. (1984) Transglutaminases. II. Lowry 0. H.. Rosebrough N. J.. Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. hiol. Chem. 193, 265-275. 12. Messer A. and Smith D. (1977) I!r t*irro behavior of granule cells from a granular neurological mutants of mice. Brain Res. 130, 13-23. II. Structure requirements of the amine 13. Pincus J. H. and Waelsch H. (196X) The specificity of transglutaminase: substratc. Arch.7 Biochern. Biophys. 126, 44-52. Quash G.. Ripoll H. and Mardon M. (1983) Differences in polyamines availability and insertion into 14. Roth A.-M.. tibronectins released from normal and transformed cells. Biochem. 1. 210, 137-144. between polyamines and nucleic acids: changes of polyamines and IS. Seiler N. and Lambert W. (1975) Interrelation nucleic acid concentrations in the developing rat brain. J. Nertrochcm. 24, S-13. B. F. (1984) Polyamines and the development of isolated neurons in cell 16. Seiler N., Sarhan S. and Roth-Schechter culture. Netrrochem. Res. 9, 871-885. E. and Sidman R. L. (1977) Histogenesis of mouse cerebellum in microwell cultures: Cell reaggregation 17. Trenkner and migration. fiber and synapse formation. J. Cell Bid. 75, 915-925. 18. Yavin Z. and Yavin E. (1980) Survival and maturation of cerebral neurons on poly (t_-lysine) surfaces in the absence of serum. Devl Biol. 75, 454-459. I.
for Neurosciences
M.
GILAD*
and Behavioral
and
Research.
VARDA
H.
GILAD*
The Weizmann
Institute
of Science.
Rehovot.
Israel
( Accqwd 22 ~ffrciz 19%) Abstract-The effects of dansylcadaverine and methylamine. competitive inhibitors of transglutaminase. were examined in primary cultures of dissociated rat ccrehcllar neurons. Addition of the drugs at pfating time resulted 24 hr later in irreversible cytotoxic effects evidenced by failure of aggregation and neurite formation. Cytotoxicity was dose-dependent with methylamine being more potent (I(‘~,,= 20 PM) than dansylcadaverine (I(‘ 5,, = 30 PM). The cytotoxic effects were less potent when drugs were added 24 hr after plating. the time when neurons had already begun to extend neurites. Drugs were effective in the various sera and heat-inactivated sera tested. We concluded that low doses of methylamine and dansylcadaverine have potent toxic effects on primary neuronal cultures. Cerehellar K(~J H*oT(/.s: Transglutaminase inhibitors. Methylamine. Cerebellum. Transglutaminasc.
cultures.
Cytotoxicity,
Monodansylcadaverine.
Monodansylcadaverine (DC) and methylamine (MA) are well-known competitive inhibitors of the enzyme transglutaminase (TG)‘.“‘. which catalyzes the covalent binding of small molecular have been weight primary amines to glutamine residues on polypeptides. ‘.I3 These inhibitors used in recent studies as indicators for TG participation in various biological functions.‘.‘.7-X Membrane associated TG activity was implicated in several developmental functions,* such as receptor-mediated endocytosisX and cell-substrate interactions (adhesion).‘.’ High TG activity was recently observed during early stages of neuronal growth in the rat cerebellum.’ In the present study therefore we sought to characterize the effects of DC and MA on dissociated rat cerebellar neurons in primary cultures, where neuronal growth and differentiation can be closely followed under controlled conditions.“-‘“.” Primary cultures were prepared from fi-day-old mechanically dissociated rat cerebellum, as reported before.” The dissociated cells were plated (400/mm2) on polylysine-coated glass coverslips” situated in 35 mm plastic Petri dishes. Drugs or drug combinations were added at plating time, or 24 hr after plating. DC and MA were purchased from Sigma Chemical Co., St Louis, MO, U.S.A. DC was dissolved in minimal volume of dimethyl sulfoxide (DMSO) and diluted to the final concentration with the culture medium. At the lowest dilution, DMSO alone (0.5% v/v) had no effect on cultures. For biochemical assays ceils were scraped off the culture dish and homogenized in 50 mM Tris-HCI buffer (pH 7.4) containing 7.5 mM dithiothreitol. TG activity was assayed as previously described.” Protein concentrations were determined according to the method of Lowry ef al.” The continuous presence of DC or MA, from plating time, prevented cell aggregation, neurite formation and caused cell death (Fig. 1, left and middle rows) in a dose-dependent manner with MA being more potent (IC~Q= 20 PM) than DC (ICY,,= 33 wM) (Fig. 2). Cytolethal effects of MA (50 PM) were observed as early as 4 hr after plating (Fig. 1, left row). No early effects of DC (50 PM) were observed, probably indicating that DC did not affect the attachment of cells to the polylysine-coated surface. The cytotoxic effects of DC and MA were not dependent on the serum used. Similar effects were observed in media containing fetal calf, rat, or human sera, which were either normal or heat-inactivated (results not shown). A~br~~iurj~~i.s: DC, monodansyi~daverine~ Prchcnt address: Veterans Administration Wars Parkway. Boston. MA 02132. U.S.A.
DN 4:5-A
MA. methylamjne; TG, transglut~lmin~l5c. Medical Center. Spinal Cord Injury Research.
401
1400 Veterans
of Foreign
402
G. M.
Glad
and
IO Amine
V. H.
Gilad
too concentration
1000
(p M)
Fig. 2. Dose-dependent effect of MA and DC on the numbers of neurcms bearing neurrtes in 24hr-old cultures. Drugs were present from plating time. Neurons were considered to hear neurites when the extensions were equal to or longer than twice the cell body size. Results are expressed as percent of total cell number per dish. Each point represents the mean value of 3 cultures in 2 separate experiments (SD. values did not exceed 20% of mean values).
The cytotoxic effects of the drugs were irreversible, as no recovery was detected after washing and changing to fresh medium not containing drugs (Fig. 1. right row). The present study demonstrates that DC and MA exert irreversible cytotoxic effects on dissociated cultured rat cerebellar neuroblasts, which are evidenced by failure of cell aggregation and neurite formation. eventually leading to cell death. MA is more potent and its effects occur earlier than DC. The effects of these drugs are less potent when added to 24- or 48-hr-old cultures containing neurons with extended neurites (Fig. 3). Previous studies with these TG inhibitors have indicated that membrane associated TG activity may participate in several developmental functions,’ such as receptor-mediated endocytosis” and cell-substratum interactions (adhesion). ‘.’ It is noteworthy, however, that the dose-dependent cytotoxic effects presently observed to occur after many hours, can be achieved with much lower concentrations than those needed to inhibit short-term processes, such as receptor-mediated endocytosis,’ where TG activity has been implicated. In the brain a substantial amount of TG activity may be associated with or even present on neuronal membranes.” TG activity in our cerebellar cultures was about 100 pmol/mg protein/hr at plating time and nearly doubled in 24”hr-old or older cultures. The demonstration that cell aggregation and neurite formation are inhibited by DC and MA. two processes which require cell-cell and cell-substratum interactions, imply that membrane associated TG activity may play a role in these processes. Yet, the adverse drug effects on growth might have been secondary to cell death, therefore the effects of sublethal doses of DC and MA on TG activity in cultures will have to be determined. Polylysine, used in this study to coat the growth surfaces, is a poor substrate for TG (G. M. Gilad and L. E. Varon, unpublished observations), but other extracellular components, such as fibronectin, a known substrate of TG,lJ may serve as substrates in such interactions. Interestingly, substantial TG activity is present in the various sera tested. Solutions containing 10% sera had average activity of 22.5 2 1.5 pmollmg protein/hr. Further, heat inactivation led only to a reduction of about 32% in serum TG activity. This finding raises the possibility that serum TG activity may participate in some aspects of cell development at least in tissue culture. Polyamines, known substrates of TG.“.“’ are present in the brain in high concentrations during and inhibition of polyamine biosynthesis in dissociated embryonic CNS cultures development,” can inhibit neuronal development.5%‘” These findings indicate that DC and MA may exert some of their effects by competing with the natural polyamines as substrates for TG catalyzed reactions. it would be interesting therefore to determine whether or not polyamines can prevent the cytotoxic effects of the drugs.
Dansylcadaverine
and methylamine
in cerebellar
cultures
403
Fig. 3. Effects of SO WM MA (h) and SO FM DC (c) on 2.bhr-old cultures. Drugs were added 24 hr after plating hy changing the medium and cultures were grown for additional 24 hr. Controls (a) were grown for 4X hr in the ahsenoc of drugs. Orlgin:ll mngnilication = x IhO. Bar = 120 FM.
Dansylcadaverine
and methylamine
in cerebellar cultures
405
In summary, MA and DC have potent toxic effects on primary cultures of rat cerebellar neurons. The primary cellular effects of these drugs are unknown. Calcium activated TG is one process which has been implicated, due to the competitive inhibition exerted on TG activity by MA and DC. Acknow/~r/~cmc,rr/s-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 Bersten A. M.. Ahkong 0. F.. Hallinan T.. Nelson S. J. and Lucy J. A. (19X3) inhibition of the formation of myotubes irr virro by inhibitors of transglutaminase. Biorhim. hiophys. Acfcr 762, 429-436. catalyzed incorporation of putrescine into surface proteins of mouse neuro2. Chen Y. K. (1984) Transglutaminase blastoma cells. Molec. cell. Biochem. 58, Yl-97. 3. Comwell M. M.. Juliano R. L. and Davies P. J. A. (1983) inhibition of the adhesion of Chinese hamsterovary cells by the naphytylsulfonamides dansylcadaverine and N-(6-aminohexyl)-5-chloro-l-naphthylenesulf~~namide (~7). Biochim. hiophys. Actor 762, 414-Il9. Anu. Ret,. Biochon. 49, 517-531. 4. Folk J. F. (19X0) Transglutaminases. 5. Gilad G. M. and Gilad V. H. (1986) Polyamines affect growth of cultured rat cerebellar neurons in different sera. Inr. J. Devl Neurosci. 4, 195-208. activity in rat brain: characterization. distribution and changes 6. Gilad G. M. and Varon L. E. (1985) Transglutaminase with age. J. Nerrrorhertr. 45, 1522-1526. V.. Schapf R. E., Hanauske-Abel H. M. and Schulte-Wissermann H. (1982) Transglutaminase and 7. Gunzler polyamine dependence of effector functions of human immunocompetent cells. The effect of specific inhibitors on lymphocyte proliferation and granulocyte chemiluminescence. FEBS Let/. 150, 3Y0-396. inhibits internalization of 8. Haigler H. T.. Maxfield F. R.. Willingham M. C. and Pastan I. (1979) Dansylcadaverine ‘“SI-epidermal growth factor in BALB 3T3 cells. J. hiol. Chem. 255, 1239-1241. 9. Kingsbury A. E.. Gallo V., Woodhams P. L. and Balazs R. (IYgS) Survival. morphology and adhesion properties of cerebellar interneurons cultured in chemically defined and serum-supplemented medium. Devl Brnirl Res. 17, 17-2.5. Mokec. cc/l. Biochem. 58, Y-3.5. IO. Lorand L. and Conrad S. M. (1984) Transglutaminases. II. Lowry 0. H.. Rosebrough N. J.. Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. hiol. Chem. 193, 265-275. 12. Messer A. and Smith D. (1977) I!r t*irro behavior of granule cells from a granular neurological mutants of mice. Brain Res. 130, 13-23. II. Structure requirements of the amine 13. Pincus J. H. and Waelsch H. (196X) The specificity of transglutaminase: substratc. Arch.7 Biochern. Biophys. 126, 44-52. Quash G.. Ripoll H. and Mardon M. (1983) Differences in polyamines availability and insertion into 14. Roth A.-M.. tibronectins released from normal and transformed cells. Biochem. 1. 210, 137-144. between polyamines and nucleic acids: changes of polyamines and IS. Seiler N. and Lambert W. (1975) Interrelation nucleic acid concentrations in the developing rat brain. J. Nertrochcm. 24, S-13. B. F. (1984) Polyamines and the development of isolated neurons in cell 16. Seiler N., Sarhan S. and Roth-Schechter culture. Netrrochem. Res. 9, 871-885. E. and Sidman R. L. (1977) Histogenesis of mouse cerebellum in microwell cultures: Cell reaggregation 17. Trenkner and migration. fiber and synapse formation. J. Cell Bid. 75, 915-925. 18. Yavin Z. and Yavin E. (1980) Survival and maturation of cerebral neurons on poly (t_-lysine) surfaces in the absence of serum. Devl Biol. 75, 454-459. I.