J. Biochem. 107, 817 820 (1990)
Uptake of 3H-Labeled l-Aminooxy-3-Aminopropane by Baby Hamster Kidney Cells1 Tapani Hyvonen,*-2 Alex R. Khomutov,** Radii M. Khomutov,** Seppo Lapinjoki,*** and Terho O. Eloranta' Departments of 'Biochemistry and '"Pharmaceutical Chemistry, University of Kuopio, P.O. Box 6, SF-70211 Kuopio, Finland; and "Institute of Molecular Biology, the U.S.S.R. Academy of Sciences, Vavilov Street 32, Moscow, 117 984 U.S.S.R Received for publication, September 26, 1989
The uptake, catabolism, and release of 3H-labeled l-aminooxy-3-aminopropane, a new putrescine analog shown to be a potent polyamine antimetabolite, into and from baby hamster kidney cells (BHK21/C13) were studied. The results show that [3H] -l-aminooxy3-aminopropane (APA) is not concentrated in the cell, does not compete with polyamines for transport and reveals no difference in uptake between polyamine-depleted and control cells. After a 12-h culture, 60% of APA was recovered intact in the culture media. At this time point, only 30% of the intracellular radioactivity was intact APA, showing that the drug is catabolized in the cells. This intracellular ratio persisted throughout the 4-day culture period. The metabolites of APA were not characterized further. The results indicate that the drug is not recognized as a polyamine by the cells and does not replace or interfere with the polyamines in cellular functions. Thus, its potent affinity to ornithine decarboxylase and spermidine synthase is likely to be due to close structural similarity with the intermediates formed in these reactions. This has implications for the mechanisms involved.
Polyamines, putrescine, spermidine, and spermine, are essential constituents of living cells and the importance of polyamines in cellular growth regulation, differentiation, and in many metabolic events is well documented {1-4). Several compounds have been tested as inhibitors of polyamine biosynthesis or interconversion (3-5). 1-Aminooxy-3-aminopropane (APA) is a new potent inhibitor of mammalian (6, 7) and bacterial (8) ornithine decarboxylase in vitro and a potent inhibitor of cell growth and of macromolecular synthesis in cultured cells (9). APA is also an irreversible inactivator of adenosylmethionine decarboxylase and a competitive inhibitor of spermidine synthase in vitro (6). APA is a putrescine analog with an H 2 N0 group, being isosterically equivalent to the H2NCH2 fragment, that confers APA reactivity toward carbonyl compounds and makes it monocationic at physiological pH. In the present paper we try to determine the functional limits of the analogy between APA and putrescine in cellular physiology.
1 This work was supported by the National Council for Natural Sciences, Finland. 2 To whom correspondence should be addressed. Abbreviation: APA, l-aminooxy-3-aminopropane.
C H
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MATERIALS AND METHODS
Synthesis of [3H]APA—[3H]APA was synthesized as follows (Scheme 1). To a solution of 40 mg of /9-ethoxyethylidene-aminooxypropionitrile (10) in 1.6 ml of ethanol, 0.4 ml of 0.5 M CoCl2-6H2O in ethanol was added. Then 40 mg of NaBH, (Serva) and 50 mCi of NaBT, (1.9 Ci/mmol, "Izotope," U.S.S.R.) in 3 ml of ethanol was added dropwise while maintaining the temperature at 25*C. After overnight stirring at 4*C, 2.5 ml of 1 M HC1 was added and the reaction mixture was evaporated in a vacuum to dryness. The residue was dissolved in water and applied to a Dowex 5 0 x 8 (H+-form) column (2.0 ml). Elution was performed subsequently with H2O, 0.5 M HC1, and 1.0 M HC1. The 1.0 M HC1 fractions containing [3H]APA were evaporated in a vacuum, and the residue was dried in a vacuum over P2O5/KOH and treated with warm isopropanol. The yield was 25 mg (61%) of radiochemically pure [3H]APA (specific radioactivity 18.7 mCi/mmol), which was proved to be identical to the noalabeled APA with the methods used (11). Cell culture and Uptake Studies—BHK21/C13, a continuous cell line derived from baby hamster kidney, was maintained as described earlier (9). The experiments were started from confluent cultures by subculturing the cells in
l.NaBT 4 /CoCl 2 C=N-O-CH2-CH2-C=N
C2H50 Vol. 107, No. 6, 1990
> 2 HC1 • H2N-O-CH2-CH2-CH2-NH2 2. HC1/H2O
Scheme 1
817
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T. Hyvonen et al.
a 24-well plate (usually 50,000 cells/1.9 cm2 well) and letting the cells adhere for 24 h before experiments. The cells were labeled (unless otherwise stated) with 100 //M [3H]APA (18.7mCi/mmol). Then, in uptake studies, the cells were washed twice with the medium without the label, and disrupted with water, and the radioactivity soluble in 5% trichloroacetic acid was counted with a liquid scintillation counter. The trichloroacetic acid-insoluble radioactivity was negligible. In efflux studies, the cells were washed after labeling as above and culturing was continued in normal medium. At the indicated time points, the media and cells were collected and the radioactivity was counted. Cell numbers were counted by using a Coulter Counter ZB (Coulter Electronics, Dunstable, Beds, U.K.) from 3 or 4 wells during uptake and efflux studies (twice during 24 h experiments in order to eliminate the effect of growth on cell number). Cell diameter and volume were measured by half-count technique using 12.92//m (in diameter) latex particles (Coulter) as a standard. Separation of APA and Its Metabolites by HPLC—The HPLC method used for determination of adenosylmethionine and decarboxylated adenosylmethionine (9) was modified to separate APA and its metabolites. A 20//I aliquot of the 10% sulfosalicylic acid-soluble portion of the cell homogenate or medium sample was applied to a reversed-phase column (Hewlett-Packard Hypersil ODS 5 nm, 100 X 2.1 mm) equilibrated for 5 min with buffer A (8 mM octanesulfonate and 0.1 mM EDTA in a mixture of 0.1 M potassium phosphate, pH 2.5, and acetonitrile, 98 : 2, v/ v). The column was then eluted with buffer A for 5 min, followed by a linear gradient to 25% buffer A, 75% buffer B (8 mM octanesulfonate in a mixture of 0.2 M potassium phosphate, pH 3.1, and acetonitrile, 7 : 3, v/v) over 15 min (i.e. 5%/min), then holding this mixture (25% buffer A, 75% buffer B) for 5 min, and back to the starting conditions over 3 min at aflowrate of 0.5 ml/min at 37*C, using an HP 1090 Liquid Chromatograph (Hewlett Packard, Waldbronn, F.R.G.). The eluate was collected in 0.5 ml fractions and counted in a liquid scintillation counter (LKB-Wallac 1214 RackBeta, Turku, Finland). The significance of differences was computed by using Student's two-tailed t test.
RESULTS
The uptake of APA into BHK21/C13 cells was concentration- and time-dependent (Figs. 1 and 2). When a 1-h labeling time was used, accumulation of radioactivity in the cells was directly proportional to the extracellular concentration of [3HJAPA (Fig. 1). With prolonged labeling at a fixed concentration (100pM) of [3H]APA, the uptake of radioactivity revealed two phases, a rapid initial one reaching a level of 250pmol/10 6 cells within 30 min followed by a slow further uptake approaching a plateau of 700 pmol/109 cells within 8 h (Fig. 2). The diameter of BHK21/ C13 cells was determined to be 16.5 ± 0.6 //m (means ± SD, n — 3) which is very close to the reported diameter of human diploid fibroblasts (12). Hence, the volume of 108 cells is about 2.5 //I. Therefore, the intracellular concentration of radioactivity was roughly equal to the extracellular one by the end of the rapid initial uptake and then slowly increased 3-fold within 8 h. Analysis of the distribution of radioactivity in HPLC fractions of the 10% sulfosalicylic acid-soluble portion of APA-labeled cells and media revealed that this drug was catabolized in the cells but not in the cell culture media. After culturing for 12 h to 4 days, 30% of the intracellular label still eluted in the same fractions as intact standard (Fig. 3; 12-hdata not shown). So, although the radioactivity in the cells eventually reached the level 3-fold the extracellular one after 8-12 h (Fig. 2), the intracellular concentration of intact APA never exceeded the concentration in the medium. In the medium, APA seems to be the major radioactive compound even after 4 days of culture indicating remarkable stability. The metabolites formed in the cells are not seen in the media analyses because the volume of the medium is about a thousandfold the cellular one. The polyamines, putrescine and spermidine, did not compete with APA for the uptake. A fivefold excess of putrescine or an equal amount of spermidine reduced the uptake of APA only by 20% (Table I). A higher concentration of spermidine was not used because the generation of toxic metabolites by serum oxidases would complicate the interpretation of the results (13, 14). Prolonged treatment
o
0
10
20
time(h) [uM] Fig. 1. Effect of drug concentration on the uptake of [*H] APA by BHK21/C13 cells. BHK21/C13 cells were grown for two days in the presence ( • ) or in the absence (o) of 5 mM ff-difluoromethylornithine before labeling with [3H]APA for 1 h. The results are means ±SD of triplicate cultures.
Fig. 2. Accumulation of APA into BHK21/C13 cells. Following the 24-h adhering period, BHK21/C13 cells were cultured in the presence of 100//M [3H]APA. At the indicated time points cells were washed twice with culture medium and disrupted with water, and the acid-soluble radioactivity was counted. The results are means±SD of triplicate cultures. When not shown, the SD bars fall within the symbols. J. Biochem.
Cellular Uptake of
0
10
20
fraction number Fig. 3. Separation of [*H]APA and of its metabolites in cells and media by HPLC. After a 2 or 4 day labeling time the cells (approximately 2-5xlO'/dish, cultured in duplicate) and the labeling media were collected, deproteinized and filtered. Then 20//I aliquots were subjected to HPLC and the radioactivities in 0.5 ml fractions of the eluate were counted. The total radioactivity levels in the 20-^1 aliquots of the standard, 48-h cells, 96-h cells, 48-h medium, and 96-h medium were 57,596, 5,653, 1,834, 38,685, and 41,830 cpm, respectively. TABLE I. Effect of poly amines on APA uptake in control and DFMO-treated BHK21/C13 cells. Cells were grown for 24 h in the presence or absence of 5 mM DFMO. The 1-h uptake was measured in the absence or presence of 500 //M putrescine or 100 //M spermidine using 100pM [JH]APA. Results are means±SD of triplicate cultures. APA uptake (pmol/10* cells/h) Control cells DFMO-treated cells No additions 655 ±69 640 ±40 500 n M putrescine 524 ± 22 505 ±50 100 //M spermidine 500±8 536 ±50
with a-difluoromethylornithine, considered to-be a specific polyamine antimetabolite, was then used to create polyamine-depleted cells. Such a treatment is known to stimulate the polyamine-uptake system of the cells (15), but had no effect on the uptake of APA (Fig. 1 and Table I). Thus, the polyamine transport mechanism is unlikely to play a role in the uptake of this drug. This result is consistent with the earlier assumption that APA does not compete with putrescine and spermidine for the polyamine transport system (9) and that the polyamine transport mechanism is unlikely to play any role in the uptake of this drug. The release of radioactivity from the cells labeled with [3H] APA was not affected by the presence of 100 //M APA or putrescine in the medium (data not shown). Nor did the 48-h pretreatment with unlabeled APA have any effect on the efflux (Fig. 4, A and B). This indicates that APA may Vol. 107, No. 6, 1990
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l-Aminooxy-3-Aminopropane
time(h) Fig. 4. Release of radioactivity from the BHK21/C13 cells into the medium after labeling for 1 h (A) or for 24 h (B) with 100 //M [*H]APA. Closed symbols represent control cells ( • ) and media ( • ) , open symbols, cells and media from cultures pretreated with 100 /iM APA for two days, respectively. Total cellular radioactivity (100%) in part A was 1,352 ± 118 cpm/well and in part B 4,197 ± 262 cpm/well, in the case of control cells, and l,307±64 and 2,959±422 cpm/weU, in the case of APA-treated cells, respectively. The results are means ± SD of triplicate cultures. The values of cellular radioactivity in the lower panel (B) differ significantly (p< 0.001) from the corresponding time point values (1-24 h) in the upper panel (A). Correspondingly, the radioactivity values of the media at the indicated time points (1-24 h) differ significantly Q>
Uptake of 3H-Labeled l-Aminooxy-3-Aminopropane by Baby Hamster Kidney Cells1 Tapani Hyvonen,*-2 Alex R. Khomutov,** Radii M. Khomutov,** Seppo Lapinjoki,*** and Terho O. Eloranta' Departments of 'Biochemistry and '"Pharmaceutical Chemistry, University of Kuopio, P.O. Box 6, SF-70211 Kuopio, Finland; and "Institute of Molecular Biology, the U.S.S.R. Academy of Sciences, Vavilov Street 32, Moscow, 117 984 U.S.S.R Received for publication, September 26, 1989
The uptake, catabolism, and release of 3H-labeled l-aminooxy-3-aminopropane, a new putrescine analog shown to be a potent polyamine antimetabolite, into and from baby hamster kidney cells (BHK21/C13) were studied. The results show that [3H] -l-aminooxy3-aminopropane (APA) is not concentrated in the cell, does not compete with polyamines for transport and reveals no difference in uptake between polyamine-depleted and control cells. After a 12-h culture, 60% of APA was recovered intact in the culture media. At this time point, only 30% of the intracellular radioactivity was intact APA, showing that the drug is catabolized in the cells. This intracellular ratio persisted throughout the 4-day culture period. The metabolites of APA were not characterized further. The results indicate that the drug is not recognized as a polyamine by the cells and does not replace or interfere with the polyamines in cellular functions. Thus, its potent affinity to ornithine decarboxylase and spermidine synthase is likely to be due to close structural similarity with the intermediates formed in these reactions. This has implications for the mechanisms involved.
Polyamines, putrescine, spermidine, and spermine, are essential constituents of living cells and the importance of polyamines in cellular growth regulation, differentiation, and in many metabolic events is well documented {1-4). Several compounds have been tested as inhibitors of polyamine biosynthesis or interconversion (3-5). 1-Aminooxy-3-aminopropane (APA) is a new potent inhibitor of mammalian (6, 7) and bacterial (8) ornithine decarboxylase in vitro and a potent inhibitor of cell growth and of macromolecular synthesis in cultured cells (9). APA is also an irreversible inactivator of adenosylmethionine decarboxylase and a competitive inhibitor of spermidine synthase in vitro (6). APA is a putrescine analog with an H 2 N0 group, being isosterically equivalent to the H2NCH2 fragment, that confers APA reactivity toward carbonyl compounds and makes it monocationic at physiological pH. In the present paper we try to determine the functional limits of the analogy between APA and putrescine in cellular physiology.
1 This work was supported by the National Council for Natural Sciences, Finland. 2 To whom correspondence should be addressed. Abbreviation: APA, l-aminooxy-3-aminopropane.
C H
3\
MATERIALS AND METHODS
Synthesis of [3H]APA—[3H]APA was synthesized as follows (Scheme 1). To a solution of 40 mg of /9-ethoxyethylidene-aminooxypropionitrile (10) in 1.6 ml of ethanol, 0.4 ml of 0.5 M CoCl2-6H2O in ethanol was added. Then 40 mg of NaBH, (Serva) and 50 mCi of NaBT, (1.9 Ci/mmol, "Izotope," U.S.S.R.) in 3 ml of ethanol was added dropwise while maintaining the temperature at 25*C. After overnight stirring at 4*C, 2.5 ml of 1 M HC1 was added and the reaction mixture was evaporated in a vacuum to dryness. The residue was dissolved in water and applied to a Dowex 5 0 x 8 (H+-form) column (2.0 ml). Elution was performed subsequently with H2O, 0.5 M HC1, and 1.0 M HC1. The 1.0 M HC1 fractions containing [3H]APA were evaporated in a vacuum, and the residue was dried in a vacuum over P2O5/KOH and treated with warm isopropanol. The yield was 25 mg (61%) of radiochemically pure [3H]APA (specific radioactivity 18.7 mCi/mmol), which was proved to be identical to the noalabeled APA with the methods used (11). Cell culture and Uptake Studies—BHK21/C13, a continuous cell line derived from baby hamster kidney, was maintained as described earlier (9). The experiments were started from confluent cultures by subculturing the cells in
l.NaBT 4 /CoCl 2 C=N-O-CH2-CH2-C=N
C2H50 Vol. 107, No. 6, 1990
> 2 HC1 • H2N-O-CH2-CH2-CH2-NH2 2. HC1/H2O
Scheme 1
817
818
T. Hyvonen et al.
a 24-well plate (usually 50,000 cells/1.9 cm2 well) and letting the cells adhere for 24 h before experiments. The cells were labeled (unless otherwise stated) with 100 //M [3H]APA (18.7mCi/mmol). Then, in uptake studies, the cells were washed twice with the medium without the label, and disrupted with water, and the radioactivity soluble in 5% trichloroacetic acid was counted with a liquid scintillation counter. The trichloroacetic acid-insoluble radioactivity was negligible. In efflux studies, the cells were washed after labeling as above and culturing was continued in normal medium. At the indicated time points, the media and cells were collected and the radioactivity was counted. Cell numbers were counted by using a Coulter Counter ZB (Coulter Electronics, Dunstable, Beds, U.K.) from 3 or 4 wells during uptake and efflux studies (twice during 24 h experiments in order to eliminate the effect of growth on cell number). Cell diameter and volume were measured by half-count technique using 12.92//m (in diameter) latex particles (Coulter) as a standard. Separation of APA and Its Metabolites by HPLC—The HPLC method used for determination of adenosylmethionine and decarboxylated adenosylmethionine (9) was modified to separate APA and its metabolites. A 20//I aliquot of the 10% sulfosalicylic acid-soluble portion of the cell homogenate or medium sample was applied to a reversed-phase column (Hewlett-Packard Hypersil ODS 5 nm, 100 X 2.1 mm) equilibrated for 5 min with buffer A (8 mM octanesulfonate and 0.1 mM EDTA in a mixture of 0.1 M potassium phosphate, pH 2.5, and acetonitrile, 98 : 2, v/ v). The column was then eluted with buffer A for 5 min, followed by a linear gradient to 25% buffer A, 75% buffer B (8 mM octanesulfonate in a mixture of 0.2 M potassium phosphate, pH 3.1, and acetonitrile, 7 : 3, v/v) over 15 min (i.e. 5%/min), then holding this mixture (25% buffer A, 75% buffer B) for 5 min, and back to the starting conditions over 3 min at aflowrate of 0.5 ml/min at 37*C, using an HP 1090 Liquid Chromatograph (Hewlett Packard, Waldbronn, F.R.G.). The eluate was collected in 0.5 ml fractions and counted in a liquid scintillation counter (LKB-Wallac 1214 RackBeta, Turku, Finland). The significance of differences was computed by using Student's two-tailed t test.
RESULTS
The uptake of APA into BHK21/C13 cells was concentration- and time-dependent (Figs. 1 and 2). When a 1-h labeling time was used, accumulation of radioactivity in the cells was directly proportional to the extracellular concentration of [3HJAPA (Fig. 1). With prolonged labeling at a fixed concentration (100pM) of [3H]APA, the uptake of radioactivity revealed two phases, a rapid initial one reaching a level of 250pmol/10 6 cells within 30 min followed by a slow further uptake approaching a plateau of 700 pmol/109 cells within 8 h (Fig. 2). The diameter of BHK21/ C13 cells was determined to be 16.5 ± 0.6 //m (means ± SD, n — 3) which is very close to the reported diameter of human diploid fibroblasts (12). Hence, the volume of 108 cells is about 2.5 //I. Therefore, the intracellular concentration of radioactivity was roughly equal to the extracellular one by the end of the rapid initial uptake and then slowly increased 3-fold within 8 h. Analysis of the distribution of radioactivity in HPLC fractions of the 10% sulfosalicylic acid-soluble portion of APA-labeled cells and media revealed that this drug was catabolized in the cells but not in the cell culture media. After culturing for 12 h to 4 days, 30% of the intracellular label still eluted in the same fractions as intact standard (Fig. 3; 12-hdata not shown). So, although the radioactivity in the cells eventually reached the level 3-fold the extracellular one after 8-12 h (Fig. 2), the intracellular concentration of intact APA never exceeded the concentration in the medium. In the medium, APA seems to be the major radioactive compound even after 4 days of culture indicating remarkable stability. The metabolites formed in the cells are not seen in the media analyses because the volume of the medium is about a thousandfold the cellular one. The polyamines, putrescine and spermidine, did not compete with APA for the uptake. A fivefold excess of putrescine or an equal amount of spermidine reduced the uptake of APA only by 20% (Table I). A higher concentration of spermidine was not used because the generation of toxic metabolites by serum oxidases would complicate the interpretation of the results (13, 14). Prolonged treatment
o
0
10
20
time(h) [uM] Fig. 1. Effect of drug concentration on the uptake of [*H] APA by BHK21/C13 cells. BHK21/C13 cells were grown for two days in the presence ( • ) or in the absence (o) of 5 mM ff-difluoromethylornithine before labeling with [3H]APA for 1 h. The results are means ±SD of triplicate cultures.
Fig. 2. Accumulation of APA into BHK21/C13 cells. Following the 24-h adhering period, BHK21/C13 cells were cultured in the presence of 100//M [3H]APA. At the indicated time points cells were washed twice with culture medium and disrupted with water, and the acid-soluble radioactivity was counted. The results are means±SD of triplicate cultures. When not shown, the SD bars fall within the symbols. J. Biochem.
Cellular Uptake of
0
10
20
fraction number Fig. 3. Separation of [*H]APA and of its metabolites in cells and media by HPLC. After a 2 or 4 day labeling time the cells (approximately 2-5xlO'/dish, cultured in duplicate) and the labeling media were collected, deproteinized and filtered. Then 20//I aliquots were subjected to HPLC and the radioactivities in 0.5 ml fractions of the eluate were counted. The total radioactivity levels in the 20-^1 aliquots of the standard, 48-h cells, 96-h cells, 48-h medium, and 96-h medium were 57,596, 5,653, 1,834, 38,685, and 41,830 cpm, respectively. TABLE I. Effect of poly amines on APA uptake in control and DFMO-treated BHK21/C13 cells. Cells were grown for 24 h in the presence or absence of 5 mM DFMO. The 1-h uptake was measured in the absence or presence of 500 //M putrescine or 100 //M spermidine using 100pM [JH]APA. Results are means±SD of triplicate cultures. APA uptake (pmol/10* cells/h) Control cells DFMO-treated cells No additions 655 ±69 640 ±40 500 n M putrescine 524 ± 22 505 ±50 100 //M spermidine 500±8 536 ±50
with a-difluoromethylornithine, considered to-be a specific polyamine antimetabolite, was then used to create polyamine-depleted cells. Such a treatment is known to stimulate the polyamine-uptake system of the cells (15), but had no effect on the uptake of APA (Fig. 1 and Table I). Thus, the polyamine transport mechanism is unlikely to play a role in the uptake of this drug. This result is consistent with the earlier assumption that APA does not compete with putrescine and spermidine for the polyamine transport system (9) and that the polyamine transport mechanism is unlikely to play any role in the uptake of this drug. The release of radioactivity from the cells labeled with [3H] APA was not affected by the presence of 100 //M APA or putrescine in the medium (data not shown). Nor did the 48-h pretreatment with unlabeled APA have any effect on the efflux (Fig. 4, A and B). This indicates that APA may Vol. 107, No. 6, 1990
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l-Aminooxy-3-Aminopropane
time(h) Fig. 4. Release of radioactivity from the BHK21/C13 cells into the medium after labeling for 1 h (A) or for 24 h (B) with 100 //M [*H]APA. Closed symbols represent control cells ( • ) and media ( • ) , open symbols, cells and media from cultures pretreated with 100 /iM APA for two days, respectively. Total cellular radioactivity (100%) in part A was 1,352 ± 118 cpm/well and in part B 4,197 ± 262 cpm/well, in the case of control cells, and l,307±64 and 2,959±422 cpm/weU, in the case of APA-treated cells, respectively. The results are means ± SD of triplicate cultures. The values of cellular radioactivity in the lower panel (B) differ significantly (p< 0.001) from the corresponding time point values (1-24 h) in the upper panel (A). Correspondingly, the radioactivity values of the media at the indicated time points (1-24 h) differ significantly Q>
