Cell Biology International Reports, Vol. 16, No. 4, 1992
349
EFFECTS OF SPERMIDINE SYNTHASE INHIBITION IN CULTURED CHICK EMBRYO FIBROBLASTS A., Caruso*, A., Pellati*, P., Bosi*, N., Arena**, G., Stahellini*.
Embriologia *Istituto di Istologia ed Universit~ di Ferrara. ** Istituto di Istologia ed Embriologia Universit~ di Sassari.
Generale. Generale.
All correspondence should be adressed to Dr. Angelo Caruso, Istituto di Istologia ed Embriologia generale, via Fossato di Mortara 64, 44100 Ferrara, ITALY. ABSTRACT
The administration of bis-cyclohexylammonium sulfate (BCHS), a spermidine synthase inhibitor, to in vitro cultures of chick embryo fibroblasts caused a decrease in cellular spermidine levels and an increase in putrescine and spermine. Cell proliferation rate and DNA synthesis were alsD inhibited. As protein synthesis did not change, it would seem that low levels of cellular spermidine inhibit cell growth depressing DNA synthesis. INTRODUCTION
The polyamines (PA) putrescine (PUT), spermidine (SPD) and spermine (SPM) are naturally occurring cations present i~-all eukaryotic cells. The exact roles that PA play in support of normal cellular functions are not well understood. The use of specific inhibitors of enzymes in t h e polyamine biosynthetic pathway has partially elucidated the role of PA in mammalian cells. When cells are depleted of their polyamines using DFMO (Metcalf at al., 1978), a specific inhibitor of ornithine decarboxylase, or AdoDATO (Pegg et al., 1982), an inhibitor of spermidine synthase, they cease to proliferate. Thus, it is evident that the polyamines play an important role in cell growth and division. This finding has led to the discovery that polyamine depletion has therapeutical effects against some neoplastic conditions (Sunkara et al., 1987, for review). In addition, polyamine depletion has been shown to induce or inhibit cell differentiation, depending on the cell type used (Heby et al., 1987, for review). These studies have provided convincing evidence that polyamines play a role not only in proliferative but also in differential processes.
0309-1651/92/040349-10/$03.00/0
© 1992 Academic PressLtd
350
Cell Biology International Reports, VoL 16, No. 4, 1992
In order to clarify better the role of spermidine in embryonic cells, we decided to study the effect of spermidine depletion in chick embryo fibroblasts. Dicyclohexylammonium sulphate (Feuerstein et ai.,1985) and dicyclohexylamine (Mitchell et al., 1985; Mattila et ai.,1984) have been found to inhibit spermidine synthase. On the other hand, Batchelor (1986) has shown that the correct names of dicyclohexylammonium sulphate and dicyclohexylamine, sold by Sigma Chemical Company, are bis-cyclohexylammonium sulphate (BCHS) and cyclohexylamine respectively. We therefore administered BCHS to primary cultures of chick embryo fibroblasts in order to define how SPD depletion affects the metabolism of embryonic cells. MATERIALS
AND METHODS
Hubbard fertilized eggs provided by the Selice Incubator (Bubano-Imola) were incubated at 38°C and 60% of relative humidity. Cell cultures. Back skin fragments were removed under sterile conditions from 7 day old chick embryos staged according to Hamburger-Hamilton Table (Hamilton, 1952), cut into small piecesand dissociated in 0.25% trypsin (DIFCO Laboratories, Detroit, MI, 1/250) in Ca ++ and M~ ++ free Hanks balanced salt solution at room temperature for 30 min. The dissociated cells were filtered through a nylon mesh, centrifuged (35 g, I0 min), then washed with phosphate buffered saline (PBS) pH 7.4 and suspended in M e d i u m 199 (GIBCO, Grand Island,NY) plus 10% foetal calf serum (FCS) and antibiotics (medium). Aliquots of cell suspension (3 x l0 s cells/cm 2) were added to plastic flask or multiwell plates (NUNC, Roskilde, Denmark) in a humidity-saturated atmosphere (5% CO2, 37°C) for 24 hrs. After 24 hrs of incubation, the medium was substituted by Medium 199 + 10% FCS (control cultures) or by Medium 199 + 10% FCS Containing I00, 200 or 400 pM BCES (treated cultures). All the cell cultures were maintained in vitro for 24 or 48 hrs. At the end of the treatment, cell viability was measured by the ability of the ceils to exclude trypan blue (Patterson, 1979). Cell growth. At the appropriate time cells were counted using crystal violet staining in 4-well culture plates in situ according to Gillies (1986). Assay of cell polyamine content. The cells were washed with PBS, detached in 0.1% trypsin in Ca ++ and Mg ++ free Hanks balanced salt solution for 10 min at 37°C, centrifuged and homogenized in 4 vol. of 0.3 M
Cell Biology International Reports, Vol. 16, No. 4, 1992
351
perchloric acid. Cellular PA were extracted and derivatized with DNS chloride as described by Stefanelli
(1986). The HPLC equipment was composed of two 420 pumps, a mixer M 491, an SFM 25 fluorescence detector, a Data System 450 (Kontron Instruments, Milano, Italy) and a 7000 injector (Rheodyne,Inc., Cotati, CA, USA). Separations were achieved with a Spherisorb S 50DS2 5-um column (250 X 4.6 mm I.D.) (Kontron). The DNS derivatives of polyamines were separated with a solvent composed of water-acetonitrile-methanol (5:3:2) as solvent A and acetonitrile-methanol (3:2) as solvent B. The sample was eluted with a linear gradient from 28 to 90% solvent B in 15 min at a flow rate of 1.0 ml/min. 3H-thymidine incorporation. Control and treated cells were pulsed with 1.0 ~Ci 3H-thymidine (20 Ci/mmol. NEN, Bad Homburg, FRG) during the last three hours of the culture period. Cells were solubilized in 0.5 M NaOH; an aliquot was precipitated with 10% TCA (30 min. 4°C), filtered on glass microfiber filters (GF/C Whatman, Maidston, England) and washed with cold 5% TCA (TCA insoluble fraction). The filters were dried and counted in 10 ml of scintillation fluid (Pico Fluor 40, Packard, Downers Grove, IL, USA) in a Packard 2425 scintillation counter. Total cell radioctivity (uptake) was measured in separate aliquots of solubilized cells counted in i0 ml of scintillation fluid. The results obtained are expressed as dpm/103cells and percent incorporation (acid insoluble fraction/total radioactivity). SH-leucine incorporation. Control and treated cells were pulsed with 1.0 ~Ci 3H-leucine (52 Ci/mmol., NEN) during the last three hours of culture periods, then processed by TCA precipitation as described for 3H-thymidine incorporation. The results obtained are expressed as dpm/103cells and percent incorporation (acid insoluble fraction/total radioactivity).
RESULTS
Polyamine levels. BCHS treatment of chick embryo cultured fibroblasts inhibited the conversion of PUT to SPD and induced the transformation of SPD to SPM (Table 1). The cellular levels of SPD decreased to about 35% of control values after 24 hrs of BCHS administration at both the concentrations used. After 48 hrs the decrease of SPD was dose-dependent. SPD content was reduced to 38% of control values in i00 uM BCHS treated cells and to 28% in 200 ~M BCHS treated cells. The increase in cellular PUT levels, up to 400% as compared to the
CeHBiology /nternat/ona/ Reports, Vo/. 16, No. 4, 1992
352
controls, was dose and t i m e - d e p e n d e n t . The i n c r e a s e in SPM content was m o r e u n i f o r m l y d i s t r i b u t e d and al wa ys h i g h e r than 200%.
TABLE 1 Polyamine levels in chick embryo fibr0blast cultures treated with BCHS for 24 an 48 hrs. putrescine
spermidine
spermine
24 hrs Control BCHS I00 ~M BCHS 200 ~[
0.79 ~ 0.09 (1oo) 1,52 ± 0.Ii (192) 2,08 i 0.19 (263)
0.94 ± 0.08 (100) 0.40 ~ 0.07 (i00) 0.34 I0.07 (36) 0.83 iO.lO (207) 0.30 10.05 (32) 1.17 ~ 0.12 (292)
0.32 ~ 0.10 (100) 0.91 i 0.=," (284) 1.38 i 0.16 (431)
1.06 i 0.13 (100) 0.57 ~ 0.09 (100) 0.41 ~ 0,06 (38) 1.43 i 0.19 (251) 0.28 i 0.04 (28) 127,0.14 (223)
48 hrs Control BCHS I00 ~M BCHS 200~d4
* n m0~es/10s cells. Means obtained fr~ two independent experiments, each in duplicate i SD. Figures in brackets are percentages of values of untreated controls.
Cell growth. As s h o w n in Fig. i, I00 ~LM BCHS did not g r e a t l y alter the rate of cell d i v i s i o n d u r i n g the first 24 hrs of treatment. However the cell number per c u l t u r e s was s t r o n g l y r e d u c e d a f t e r 48 hrs. 200 n M BCHS r e d u c e d cell p r o l i f e r a t i o n rate b ot h after 24 and 48 hrs of treatment. Cell n u m b e r per c u l t u r e s was a lw ay s lower as c o m p a r e d ~o 100 uM BCHS t r e a t e d cells. a-H thymidine incorporation. BCH S t r e a t m e n t decreased the total 3-H t h y m i d i n e i n c o r p o r a t e d by the cells in culture (Table 2). A f t e r 24 hours, the radioactivity decreased to 83% and 64%, respectively, in i00 n M and 200 ~,M BCHS t r e a t e d cells, as c o m p a r e d w i t h the control. T h e s e values d r o p p e d to 50% and 18% of the control after 48 hours of treatment. On the other hand, the p e r c e n t a g e of 3-H t h y m i d i n e i n c o r p o r a t e d into the n e w l y s y n t h e s i z e d DNA did not c h a n g e in t r e a t e d cells as c o m p a r e d w i t h the control, b o t h after 24 and 48 h o ur s of treatment.
Cell Biology lnternational Reports, Vol. 16, No. 4, 1992
353
1.3
tO
IO X V L--
n
E :3 t-
0.7 JL
m m
U
0.4 I
I
t
0
24
48
hours Fig. I. Effect of BCHS on the growth of fibroblasts. • control, • 100pH BCHS, • The experiments were done four times results. Data shown are from a single experiment.
chick embryo 200jJM BCHS. with similar representative
3-H leucine incorporation. BCHS treatment did not interfere with either total leucine incorporation or the percentage of leuci~e incorporated into the proteins (Table 3).
Cell Biology International Reports, Vol. 16, No. 4, 1992
354 TABLE 2
mHTthymidine incorporation in chick ~ r y o fihrobl~t cultur~ treated with BCES for 24 and 48 hrs. ~H-thymidine was pulsed during the last 3 hrs. 24 hrs.
48 hrs. P
Control BCES 100~M BCES 200~uM
P
499,581 i 29,1!3 57§ 413,590 121,281
349
EFFECTS OF SPERMIDINE SYNTHASE INHIBITION IN CULTURED CHICK EMBRYO FIBROBLASTS A., Caruso*, A., Pellati*, P., Bosi*, N., Arena**, G., Stahellini*.
Embriologia *Istituto di Istologia ed Universit~ di Ferrara. ** Istituto di Istologia ed Embriologia Universit~ di Sassari.
Generale. Generale.
All correspondence should be adressed to Dr. Angelo Caruso, Istituto di Istologia ed Embriologia generale, via Fossato di Mortara 64, 44100 Ferrara, ITALY. ABSTRACT
The administration of bis-cyclohexylammonium sulfate (BCHS), a spermidine synthase inhibitor, to in vitro cultures of chick embryo fibroblasts caused a decrease in cellular spermidine levels and an increase in putrescine and spermine. Cell proliferation rate and DNA synthesis were alsD inhibited. As protein synthesis did not change, it would seem that low levels of cellular spermidine inhibit cell growth depressing DNA synthesis. INTRODUCTION
The polyamines (PA) putrescine (PUT), spermidine (SPD) and spermine (SPM) are naturally occurring cations present i~-all eukaryotic cells. The exact roles that PA play in support of normal cellular functions are not well understood. The use of specific inhibitors of enzymes in t h e polyamine biosynthetic pathway has partially elucidated the role of PA in mammalian cells. When cells are depleted of their polyamines using DFMO (Metcalf at al., 1978), a specific inhibitor of ornithine decarboxylase, or AdoDATO (Pegg et al., 1982), an inhibitor of spermidine synthase, they cease to proliferate. Thus, it is evident that the polyamines play an important role in cell growth and division. This finding has led to the discovery that polyamine depletion has therapeutical effects against some neoplastic conditions (Sunkara et al., 1987, for review). In addition, polyamine depletion has been shown to induce or inhibit cell differentiation, depending on the cell type used (Heby et al., 1987, for review). These studies have provided convincing evidence that polyamines play a role not only in proliferative but also in differential processes.
0309-1651/92/040349-10/$03.00/0
© 1992 Academic PressLtd
350
Cell Biology International Reports, VoL 16, No. 4, 1992
In order to clarify better the role of spermidine in embryonic cells, we decided to study the effect of spermidine depletion in chick embryo fibroblasts. Dicyclohexylammonium sulphate (Feuerstein et ai.,1985) and dicyclohexylamine (Mitchell et al., 1985; Mattila et ai.,1984) have been found to inhibit spermidine synthase. On the other hand, Batchelor (1986) has shown that the correct names of dicyclohexylammonium sulphate and dicyclohexylamine, sold by Sigma Chemical Company, are bis-cyclohexylammonium sulphate (BCHS) and cyclohexylamine respectively. We therefore administered BCHS to primary cultures of chick embryo fibroblasts in order to define how SPD depletion affects the metabolism of embryonic cells. MATERIALS
AND METHODS
Hubbard fertilized eggs provided by the Selice Incubator (Bubano-Imola) were incubated at 38°C and 60% of relative humidity. Cell cultures. Back skin fragments were removed under sterile conditions from 7 day old chick embryos staged according to Hamburger-Hamilton Table (Hamilton, 1952), cut into small piecesand dissociated in 0.25% trypsin (DIFCO Laboratories, Detroit, MI, 1/250) in Ca ++ and M~ ++ free Hanks balanced salt solution at room temperature for 30 min. The dissociated cells were filtered through a nylon mesh, centrifuged (35 g, I0 min), then washed with phosphate buffered saline (PBS) pH 7.4 and suspended in M e d i u m 199 (GIBCO, Grand Island,NY) plus 10% foetal calf serum (FCS) and antibiotics (medium). Aliquots of cell suspension (3 x l0 s cells/cm 2) were added to plastic flask or multiwell plates (NUNC, Roskilde, Denmark) in a humidity-saturated atmosphere (5% CO2, 37°C) for 24 hrs. After 24 hrs of incubation, the medium was substituted by Medium 199 + 10% FCS (control cultures) or by Medium 199 + 10% FCS Containing I00, 200 or 400 pM BCES (treated cultures). All the cell cultures were maintained in vitro for 24 or 48 hrs. At the end of the treatment, cell viability was measured by the ability of the ceils to exclude trypan blue (Patterson, 1979). Cell growth. At the appropriate time cells were counted using crystal violet staining in 4-well culture plates in situ according to Gillies (1986). Assay of cell polyamine content. The cells were washed with PBS, detached in 0.1% trypsin in Ca ++ and Mg ++ free Hanks balanced salt solution for 10 min at 37°C, centrifuged and homogenized in 4 vol. of 0.3 M
Cell Biology International Reports, Vol. 16, No. 4, 1992
351
perchloric acid. Cellular PA were extracted and derivatized with DNS chloride as described by Stefanelli
(1986). The HPLC equipment was composed of two 420 pumps, a mixer M 491, an SFM 25 fluorescence detector, a Data System 450 (Kontron Instruments, Milano, Italy) and a 7000 injector (Rheodyne,Inc., Cotati, CA, USA). Separations were achieved with a Spherisorb S 50DS2 5-um column (250 X 4.6 mm I.D.) (Kontron). The DNS derivatives of polyamines were separated with a solvent composed of water-acetonitrile-methanol (5:3:2) as solvent A and acetonitrile-methanol (3:2) as solvent B. The sample was eluted with a linear gradient from 28 to 90% solvent B in 15 min at a flow rate of 1.0 ml/min. 3H-thymidine incorporation. Control and treated cells were pulsed with 1.0 ~Ci 3H-thymidine (20 Ci/mmol. NEN, Bad Homburg, FRG) during the last three hours of the culture period. Cells were solubilized in 0.5 M NaOH; an aliquot was precipitated with 10% TCA (30 min. 4°C), filtered on glass microfiber filters (GF/C Whatman, Maidston, England) and washed with cold 5% TCA (TCA insoluble fraction). The filters were dried and counted in 10 ml of scintillation fluid (Pico Fluor 40, Packard, Downers Grove, IL, USA) in a Packard 2425 scintillation counter. Total cell radioctivity (uptake) was measured in separate aliquots of solubilized cells counted in i0 ml of scintillation fluid. The results obtained are expressed as dpm/103cells and percent incorporation (acid insoluble fraction/total radioactivity). SH-leucine incorporation. Control and treated cells were pulsed with 1.0 ~Ci 3H-leucine (52 Ci/mmol., NEN) during the last three hours of culture periods, then processed by TCA precipitation as described for 3H-thymidine incorporation. The results obtained are expressed as dpm/103cells and percent incorporation (acid insoluble fraction/total radioactivity).
RESULTS
Polyamine levels. BCHS treatment of chick embryo cultured fibroblasts inhibited the conversion of PUT to SPD and induced the transformation of SPD to SPM (Table 1). The cellular levels of SPD decreased to about 35% of control values after 24 hrs of BCHS administration at both the concentrations used. After 48 hrs the decrease of SPD was dose-dependent. SPD content was reduced to 38% of control values in i00 uM BCHS treated cells and to 28% in 200 ~M BCHS treated cells. The increase in cellular PUT levels, up to 400% as compared to the
CeHBiology /nternat/ona/ Reports, Vo/. 16, No. 4, 1992
352
controls, was dose and t i m e - d e p e n d e n t . The i n c r e a s e in SPM content was m o r e u n i f o r m l y d i s t r i b u t e d and al wa ys h i g h e r than 200%.
TABLE 1 Polyamine levels in chick embryo fibr0blast cultures treated with BCHS for 24 an 48 hrs. putrescine
spermidine
spermine
24 hrs Control BCHS I00 ~M BCHS 200 ~[
0.79 ~ 0.09 (1oo) 1,52 ± 0.Ii (192) 2,08 i 0.19 (263)
0.94 ± 0.08 (100) 0.40 ~ 0.07 (i00) 0.34 I0.07 (36) 0.83 iO.lO (207) 0.30 10.05 (32) 1.17 ~ 0.12 (292)
0.32 ~ 0.10 (100) 0.91 i 0.=," (284) 1.38 i 0.16 (431)
1.06 i 0.13 (100) 0.57 ~ 0.09 (100) 0.41 ~ 0,06 (38) 1.43 i 0.19 (251) 0.28 i 0.04 (28) 127,0.14 (223)
48 hrs Control BCHS I00 ~M BCHS 200~d4
* n m0~es/10s cells. Means obtained fr~ two independent experiments, each in duplicate i SD. Figures in brackets are percentages of values of untreated controls.
Cell growth. As s h o w n in Fig. i, I00 ~LM BCHS did not g r e a t l y alter the rate of cell d i v i s i o n d u r i n g the first 24 hrs of treatment. However the cell number per c u l t u r e s was s t r o n g l y r e d u c e d a f t e r 48 hrs. 200 n M BCHS r e d u c e d cell p r o l i f e r a t i o n rate b ot h after 24 and 48 hrs of treatment. Cell n u m b e r per c u l t u r e s was a lw ay s lower as c o m p a r e d ~o 100 uM BCHS t r e a t e d cells. a-H thymidine incorporation. BCH S t r e a t m e n t decreased the total 3-H t h y m i d i n e i n c o r p o r a t e d by the cells in culture (Table 2). A f t e r 24 hours, the radioactivity decreased to 83% and 64%, respectively, in i00 n M and 200 ~,M BCHS t r e a t e d cells, as c o m p a r e d w i t h the control. T h e s e values d r o p p e d to 50% and 18% of the control after 48 hours of treatment. On the other hand, the p e r c e n t a g e of 3-H t h y m i d i n e i n c o r p o r a t e d into the n e w l y s y n t h e s i z e d DNA did not c h a n g e in t r e a t e d cells as c o m p a r e d w i t h the control, b o t h after 24 and 48 h o ur s of treatment.
Cell Biology lnternational Reports, Vol. 16, No. 4, 1992
353
1.3
tO
IO X V L--
n
E :3 t-
0.7 JL
m m
U
0.4 I
I
t
0
24
48
hours Fig. I. Effect of BCHS on the growth of fibroblasts. • control, • 100pH BCHS, • The experiments were done four times results. Data shown are from a single experiment.
chick embryo 200jJM BCHS. with similar representative
3-H leucine incorporation. BCHS treatment did not interfere with either total leucine incorporation or the percentage of leuci~e incorporated into the proteins (Table 3).
Cell Biology International Reports, Vol. 16, No. 4, 1992
354 TABLE 2
mHTthymidine incorporation in chick ~ r y o fihrobl~t cultur~ treated with BCES for 24 and 48 hrs. ~H-thymidine was pulsed during the last 3 hrs. 24 hrs.
48 hrs. P
Control BCES 100~M BCES 200~uM
P
499,581 i 29,1!3 57§ 413,590 121,281
