JOURNAL OF CELLULAR PHYSIOLOGY 143:460-467 (1990)
Regulation of Polyamine Transport in Chinese Hamster Ovary Cells TIMOTHY 1. BYERS AND ANTHONY E. P E W * Departments of Physiology and Pharmacology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania I7033 Control Chinese hamster ovary (CHO) cells and mutant C H O cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyarnines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with a-difluoromethylornithine in C H O cells or via polyamine starvation of CHODCcells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyarnine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyarnine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyarnine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl1polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guany1hydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.
Polyamine levels in mammalian cells are highly regulated (reviewed by Pegg and McCann, 1982; Pegg, 1986,1988;Tabor and Tabor, 1984).It has been generally believed that this regulation takes place primarily by means of changes in the activity of key enzymes involved in the biosynthesis and metabolism of the polyamines and the activity of these enzymes show striking changes in response to perturbations of the polyamine pools. However, an increasing body of evidence suggests that the transport of polyamines across the cell membrane may also be subject to regulation and changes in the uptake or efflux of polyamines provide an additional mechanism to control and modify intracellular polyamine contents (Alhonen-Hongisto et al., 1980;Janne et al., 1983;Rinehart and Chen, 1984; Feige et al., 1986; Wallace, 1987;Kakinuma et al., 1988;Gawel-Thompson and Green, 1988,1989;Pegg et al., 1989;Kumagi et al., 1989). The important discovery by Alhonen-Hongisto et al. (1980)that treatment of cells with a-difluoromethylornithine (DFMO) leads to an increased activity of the polyamine uptake system has now been confirmed and extended in many different cell types (Janne et al., 1983;Porter and Janne, 1987;Kakinuma et al., 1988; Byers and Pegg, 1989;De Smedt et al., 1989;GawelThompson and Green, 1989).Since DFMO is a potent inhibitor of ornithine decarboxylase and leads to a depletion of the intracellular content of putrescine and spermidine (Pegg and McCann, 1982)the most plausi0 1990 WILEY-LISS. INC
ble explanation of these results is that the activity of the transport system responds to the intracellular polyamine content, but this response had not been studied in detail. In the present paper we have used Chinese hamster ovary (CHO)cells and mutant CHO cells lacking ornithine decarboxylase activity [CHODC- cells1 (Steglich and Scheffler, 1982)to investigate the regulation of polyamine uptake by intracellular polyamine concentrations and the physiological significance of this regulation.
MATERIALS AND METHODS Materials MGBG was obtained from the Aldrich Chemical Company (Milwaukee, WI). DFMO was a generous gift from Merrell Dow Research Institute (Cincinnati, OH). The methylglyoxal bis[14Cl(guanylhydrazone),[l,4l4C1putrescine, and [1,4-14Clsperminewere purchased from Amersham-Searle. Polyamine analogues synthesized as described by Bergeron et al. (1988)were generously provided by Dr. R. J. Bergeron, Department of
Received December 4, 1989; accepted February 14, 1990. *To whom reprint requestsicorrespondence should be addressed. Timothy L. Byers’s present address is Merrell Dow Research Institute, Cincinnati, OH 45215.
REGULATION OF POLYAMINE TRANSPORT
Medicinal Chemistry, University of Florida, Gainesville.
461
Cells were grown for 48-72 h, washed three times with 10 ml of 4°C PBS, and then harvested in 100-300 p1 10% trichloroacetic acid. These samples were stored at Methods 4°C for 12-24 h, sonicated for 5 min, and the proteins Cell culture. Chinese hamster ovary (CHO) cells precipitated by centrifugation. The supernatant was were grown as monolayers in a-MEM (Gibco)with 10% filtered through 0.2 pm filters (Bioanalytical Systems fetal bovine serum (FBS), 12 pg/ml penicillin, and 12 Inc.). The protein pellet was redissolved in 0.1 N NaOH pg/ml streptomycin at 37°C in 10% CO, and 93% hu- and quantitated by the method of Bradford. Polymidity. The clone C55.7 CHO cells deficient in ODC amines in the filtered supernatant were separated and activity (CHODC- cells) were graciously furnished by quantitated using a reverse phase HPLC system as deDr. C. Steglich of the University of California, Depart- scribed. ment of Biology. These cells are polyamine auxotrophs The reverse phase chromatography system utilized a because of their inability to synthesize putrescine HPLC apparatus from Beckman Instruments. Samples (Steglich and Scheffler, 1982). They were maintained were injected onto the system by a SpectraPhysics Sf in Dulbecco’s MEM containing 35 mM NaHCO,, 1.0 P8270 autosampler and passed through a Brownlee mM aminoguanidine, 12 pg/ml penicillin, and 12 pg/ml Labs RP-300 pre-column and onto a Beckman Ultrastreptomycin and supplemented with 10% Serum Plus sphere Ion Pairing 5 pm C,, (250 mm x 4.6 mm) colmedia supplement (Hazleton-Dutchland Inc), 350 pM umn at 36°C. Two buffers were used to form an acetoproline, and 0.5 M putrescine. The cells were grown as nitrile/methanol gradient. Buffer A contained 0.1 M monolayers at 37°C in 10% CO, and 93% humidity. sodium acetate (pH 4.5) and 9.1 mM octane sulfonate. Polyamine uptake experiments. The cells were Buffer B contained 0.2M sodium acetate (pH 4.51, plated in multiwell plates. After the indicated growth 9.1 mM octane sulfonate, 21% acetonitrile, and 9% period, the growth media was aspirated off and a-MEM methanol. There was one gradient step, a change from (without FBS) containing labeled uptake substrate was 30% Buffer B to 100% Buffer B in 35 min. Buffer B was added. The cells were then incubated at 37°C in 10% maintained a t 100%for 17 min. After the gradient was COz and 93% humidity. After the appropriate incuba- completed, the column was returned to initial condition time, the media was aspirated off and the cells tions in 2 min and equilibrated for 10 min before the washed twice with 1ml ice-cold a-MEM (without FBS) next run. The flow rate was 1 mumin. Detection of containing 20 mM unlabeled substrate and then twice polyamines was accomplished using a fluorescence dewith 1ml ice-cold phosphate buffered saline (PBS). The tector after a post column derivitization with owashed cells were then dissolved by sonication in 0.1 N phthalaldehyde/2-mercaptoethanol (Seiler and KnNaOH and aliquots of this solution were used for pro- odgen, 1985). The reagent was delivered a t 1ml/min in tein quantitation and for determination of radioactiv- solution prepared as follows: 0.05% (w/v) o-phthalaldeity. Protein was measured by the dye binding method hyde (Pierce, Fluka) in 0.4 M boric acid (pH 10.4) conof Bradford (1976) using the dye from Bio Rad. Radio- taining 1%methanol, 0.45% 2-mercaptoethanol, and activity was determined using a Beckman LS-3133T 0.03% (w/v) BRIJ. Fluorescence was activated a t scintillation counter after the addition of 10 ml of ACS 345 nm, and fluorescent intensity measured at I1 (Amersham-Searle). Uptake measurements were 255 nm. Fluorescent emissions were recorded and incarried out under conditions in which uptake was tegrated by a SpectraiPhysics SiP4270 integrator. Eluknown to be linear with respect to time. Where uptake tion times were 27 rnin for putrescine, 42 min for sperwas measured over a time course, the rates were de- midine, and 52 min for spermine. Commercially termined from the slopes of graphs of uptake against available polyamine hydrochlorides were used as stantime obtained by a linear regression analysis to obtain dards. the best fit line. Where the effect of DFMO on polyRESULTS amine uptake activity was quantitated, cells were exposed to 5.0 mM DFMO for 48-72 h prior to uptake The response of the polyamine transport system in measurements. Although the uptake measurements CHO cells to excess intracellular polyamine concentrawere made in a-MEM medium, our previous studies tions was examined by incubation in media containing have shown that similar transport rates were obtained polyamines. This resulted in depressed polyamine upwhen uptake was measured in PBS (Byers and Pegg, take activity (Table 1). A similar response has been reported in mouse neuroblastoma cells (Rinehart and 1989). The polyamine content in CHO and CHODC- cells Chen, 1984). Repression of polyamine transport capacwas elevated by incubation with exogenous poly- ity did not require protein synthesis since cyclohexiamines. These cells were washed extensively with PBS mide did not prevent the effect (Table 1).Since expo(37°C)prior to any measurements of radiolabeled poly- sure of CHO cells to cycloheximide for 12 h did not amine uptake. Polyamine levels were reduced in alter the rate of polyamine transport, the proteins inCHODC- cells by thoroughly washing with PBS (37°C) volved do not appear to turn over rapidly (Table 1). prior to harvesting, replating, and growth in media The reversal of the repression of the polyamine lacking polyamines. Although CHOCD- cells were transport system was examined by removing CHO cells grown in Dulbecco’s MEM, polyamine uptake was de- from an exogenous polyamine source (Fig. 1).CHO termined by incubating these cells in a-MEM, without cells maintained in media containing polyamines exFBS, containing radiolabeled substrate. hibited depressed polyamine transport activity. ReSeparation and quantitation of polyamines. The placement of this media with fresh media lacking polyintracellular polyamine content of CHO and CHODC- amines resulted in a time dependent increase in cells was determined using the following procedures. putrescine (Fig. 1A) and spermine (Fig. 1B) uptake.
462
BYERS AND PEGG
TABLE 1. Response of polyamine transport to exogenous polyamines and to cycloheximide'
Treatment None None Cycloheximide, 10 pgiml 50 pM putrescine 5 pM spermine Cycloheximide, 10 pgiml + 50 LLMuutrescine 5 LLMsnermine
+
Transport activity (nmolimg proteidh) Putrescine Spermine 3.3 t 0 . 3 5.3 ? 0.6 4.1 ? 0.2 4.6 ? 0.6 3.0 i 0.2 3.5 t 0.2 0.3 ? 0.2 0.9 i 0.2
Time (h) 0 12 12 12
+
10.3
12
0.5
?
0.4
'CHO cells were plated in the presence of 2 mM aminoguanidine a t 3 x lo4 cells/ml/well. At 72 h putrcscine, spermine, and cyclohcximide were added to the appropriate wells to give 50 p M , 5 pM,and 10 pgiml concentrations, respectively. Determination of putrescine and spermine uptake was carried out a t this time and 12 h later as indicated. The results reported are the means S.D. for 3 observations.
*
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Hours
g
o
6
,
I
12
,
I
18
,
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24
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Fig. 1. Effect of removal of an exogenous polyamine source on polyamine uptake in CHO cells. CHO cells were grown in a-MEM containing no additions (*); 2.0 mM aminoguanidine, 50 pM putrescine, and 5 pM spermine (m); or 2.0 mM aminoguanidine, 50 pM putrescine, 5 pM spermine and 5 mM DFMO (A) for 48 h. At the end of this time, the cells were washed and media added back containing no additions
(Control, *), 2.0 mM aminoguanidine (-PA, m) or 2.0 mM aminoguanidine and 5 mM DFMO (-PA+ DFMO, A). After incubation for the indicated periods, the media was removed and putrescine (A) or spermine (B) transport was measured. These values are the means * S.D. for 3 observations.
Transport activity rose to levels slightly above those in cells not exposed to polyamines and then fell to the expected control values. The 6 h peak in control cell polyamine uptake may have been in response to the addition of fresh serum as reported in other cell types (Pohjanpelto, 1976; DiPasquale et al., 1978; Wallace and Keir, 1981). The presence of DFMO did not alter the rate of response to polyamine removal, but did extend and magnify the increase in polyamine transport activity past the peak observed in untreated cells. This increase may be explained by a fall in intracellular polyamines below control levels, due to the inhibition of putrescine synthesis. Protein synthesis was mandatory for the activation of polyamine transport. The addition of cycloheximide a t the time of exogenous polyamine removal prevented the rise in polyamine transport activity observed in cells not exposed to this protein synthesis inhibitor (Fig. 2). The effect of cycloheximide exposure on the activation of polyamine transport was not due to decreased viability or cell death. CHO cells maintained in the presence of polyamines until the addition of cycloheximide had low polyamine uptake activity over a 12 h period. However, removal of this drug a t 12 h resulted in a rise in polyamine uptake equivalent to that
observed in cells not exposed to cycloheximide (data not shown). The inability of CHODC- cells to produce putrescine was exploited to deplete polyamine content without the use of DFMO. Polyamine starvation of CHODC- cells resulted in reduced putrescine and spermidine levels and a n increase in putrescine and spermine uptake which was maintained for up to 72 h and was not amplified by the presence of DFMO. In a typical experiment, growth of CHODC- cells in the absence of polyamines for 72 h resulted in putrescine and spermine uptake values of 7.0 2 0.24 and 5.6 It_ 0.6 nmollmgi protein/h, respectively; cells starved in the presence of DFMO had the respective values of 6.6 k 0.4 and 7.2 +0.6 nmol/mg protein/h. As shown in Figure 3, re-feeding polyamine starved CHODC- cells with exogenous putrescine (Fig. 3A) or spermine (Fig. 3B) resulted in a n immediate increase in the respective intracellular polyamine content and a time dependent decrease in polyamine transport activity. Within 6 h, the transport capacity for either putrescine or spermine was maximally reduced. The total intracellular level of the added polyamine declined rapidly following the repression of transport (Fig. 3). The treatment with putrescine, which elevates intra-
463
REGULATION OF POLYAMINE TRANSPORT
4.0i /
-Cycle.
2.0L4L 3.0
1 .o
0.0 0
0
8
4
12
12 t Cyclo.
8
4
Hours
Hours Fig. 2. Effect of inhibition of protein synthesis on increase in polyamine transport after removal of a n exogenous polyamine source from CHO cells. CHO cells were grown in a-MEM containing 2.0 mM aminoguanidine, 50 p M putrescine, and 5 p M spermine for 48 h. At the end of this time, the cells were washed and media added back con-
taining 2 mM aminoguanidine (FCyclo, 0 ) or 2.0 mM aminoguanidine and 10 pg/ml cycloheximide ( + Cyclo, m). After incubation for the indicated periods, the media was removed and putrescine (A)or spermine (B) transport was measured. These values are the means f S.D. for 3 observations.
8.0
- 140
7.0
-120
0,
-5m -m Y
6.0
-100
5.0 4.0
v
-80
-
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3.0 ._ 2.0 1.0-
-20
'C
-0
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3 a,
2.0 1.o
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0.0
L
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-60
a
3.0
g
-40 C
I
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2
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6
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Hours
Fig. 3. Effect of putrescine and spermine exposure on the polyamine transport activity and the polyamine profile of C H O D C cells previously starved for polyamines. CHODC- cells were plated in 35 mm dishes and grown in Dulbecco's MEM in the absence of any polyamine. At 72 h the cells contained putrescine or spermidine levels of C1.5 n m o l h g protein and the spermine content was 9.3 i 1.0 nmoli
mg. At this point, cultures were exposed to 500 p M putrescine (A) or 10 pM spermine (B).After the indicated times, cells were washed 3 X with 5 ml of PBS and harvested for polyamine analysis ( 0 , putrescine; A, spermidine; spermine) or for measurement of transport of putrescine ( 0 )or spermine (0). The values above are the means 2 S.D. for 3 dishes.
cellular spermidine and spermine as well as putrescine, led to an equally rapid decline in putrescine and spermine transport (Fig. 3A). However, exposure to exogenous spermine which increases only spermine seemed to have a somewhat more rapid effect on putrescine transport (Fig. 3B). The addition of a range of putrescine concentrations to polyamine starved CHODC- cells and measurement of the polyamine transport capacity and intracellular polyamine levels 1.5 and 3 h later confirmed that there was a decrease in polyamine transport which was dependent on both time and the accumulation of intracellular polyamines (Table 2). Cells exposed for 1.5 h to extracellular putrescine concentrations above the K, for putrescine uptake, which is about 4.5 pM (Byers et al., 1989), showed elevated polyamine levels and depressed polyamine transport activity. In contrast, CHODC- cells incubated with only 1 pM putrescine had only slightly elevated polyamine contents and did
not change transport activity. However, CHODC- cells exposed to 5 pM putrescine for 1.5 h also exhibited almost control uptake activity despite high intracellular polyamine levels. These results suggest that intracellular polyamine content must reach a threshold after which transport decreases via a time dependent mechanism. Presumably, the polyamine levels in cells exposed to putrescine concentrations allowing for the maximal rate of uptake rapidly reached this threshold level and the effects of these levels on transport are evident by 1.5 h. Cells exposed to 5 pM putrescine may have taken longer to accumulate the threshold level of polyamines, as evidenced by the persistence of transport activity at 1.5 h. However, by 3 h, the transport activity in these cells was also decreased (Table 2). The cells exposed to only 1 pM putrescine showed only a small reduction on transport capacity at 3 h by which time intracellular polyamines had increased substantially (Table 2).
.,
464
BYERS AND PEGG
TABLE 2.The effect of incubating cells previously starved for polyamines with various concentrations of exogenous putrescine on Dolvamine transDort activity and intracellular Dolvamine levels' Exogenous putrescine (pM) 0 50 10 5 1 50 10 5 1
Time (h) 0 1.5 1.5 1.5 1.5 3.0 3.0
3.0 3.0
Transport activity (nmolimg protein/h) Putrescine Spermine 7.3 ? 0.6 7.3 2 0.9 1.0 f 0.3 2.7 2 0.3 3.8 f 0.5 2.9 0.4 5.4 0.7 5.0 f 0.9 5.8 ? 0.4 4.7 ? 0.8
Regulation of Polyamine Transport in Chinese Hamster Ovary Cells TIMOTHY 1. BYERS AND ANTHONY E. P E W * Departments of Physiology and Pharmacology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania I7033 Control Chinese hamster ovary (CHO) cells and mutant C H O cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyarnines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with a-difluoromethylornithine in C H O cells or via polyamine starvation of CHODCcells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyarnine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyarnine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyarnine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl1polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guany1hydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.
Polyamine levels in mammalian cells are highly regulated (reviewed by Pegg and McCann, 1982; Pegg, 1986,1988;Tabor and Tabor, 1984).It has been generally believed that this regulation takes place primarily by means of changes in the activity of key enzymes involved in the biosynthesis and metabolism of the polyamines and the activity of these enzymes show striking changes in response to perturbations of the polyamine pools. However, an increasing body of evidence suggests that the transport of polyamines across the cell membrane may also be subject to regulation and changes in the uptake or efflux of polyamines provide an additional mechanism to control and modify intracellular polyamine contents (Alhonen-Hongisto et al., 1980;Janne et al., 1983;Rinehart and Chen, 1984; Feige et al., 1986; Wallace, 1987;Kakinuma et al., 1988;Gawel-Thompson and Green, 1988,1989;Pegg et al., 1989;Kumagi et al., 1989). The important discovery by Alhonen-Hongisto et al. (1980)that treatment of cells with a-difluoromethylornithine (DFMO) leads to an increased activity of the polyamine uptake system has now been confirmed and extended in many different cell types (Janne et al., 1983;Porter and Janne, 1987;Kakinuma et al., 1988; Byers and Pegg, 1989;De Smedt et al., 1989;GawelThompson and Green, 1989).Since DFMO is a potent inhibitor of ornithine decarboxylase and leads to a depletion of the intracellular content of putrescine and spermidine (Pegg and McCann, 1982)the most plausi0 1990 WILEY-LISS. INC
ble explanation of these results is that the activity of the transport system responds to the intracellular polyamine content, but this response had not been studied in detail. In the present paper we have used Chinese hamster ovary (CHO)cells and mutant CHO cells lacking ornithine decarboxylase activity [CHODC- cells1 (Steglich and Scheffler, 1982)to investigate the regulation of polyamine uptake by intracellular polyamine concentrations and the physiological significance of this regulation.
MATERIALS AND METHODS Materials MGBG was obtained from the Aldrich Chemical Company (Milwaukee, WI). DFMO was a generous gift from Merrell Dow Research Institute (Cincinnati, OH). The methylglyoxal bis[14Cl(guanylhydrazone),[l,4l4C1putrescine, and [1,4-14Clsperminewere purchased from Amersham-Searle. Polyamine analogues synthesized as described by Bergeron et al. (1988)were generously provided by Dr. R. J. Bergeron, Department of
Received December 4, 1989; accepted February 14, 1990. *To whom reprint requestsicorrespondence should be addressed. Timothy L. Byers’s present address is Merrell Dow Research Institute, Cincinnati, OH 45215.
REGULATION OF POLYAMINE TRANSPORT
Medicinal Chemistry, University of Florida, Gainesville.
461
Cells were grown for 48-72 h, washed three times with 10 ml of 4°C PBS, and then harvested in 100-300 p1 10% trichloroacetic acid. These samples were stored at Methods 4°C for 12-24 h, sonicated for 5 min, and the proteins Cell culture. Chinese hamster ovary (CHO) cells precipitated by centrifugation. The supernatant was were grown as monolayers in a-MEM (Gibco)with 10% filtered through 0.2 pm filters (Bioanalytical Systems fetal bovine serum (FBS), 12 pg/ml penicillin, and 12 Inc.). The protein pellet was redissolved in 0.1 N NaOH pg/ml streptomycin at 37°C in 10% CO, and 93% hu- and quantitated by the method of Bradford. Polymidity. The clone C55.7 CHO cells deficient in ODC amines in the filtered supernatant were separated and activity (CHODC- cells) were graciously furnished by quantitated using a reverse phase HPLC system as deDr. C. Steglich of the University of California, Depart- scribed. ment of Biology. These cells are polyamine auxotrophs The reverse phase chromatography system utilized a because of their inability to synthesize putrescine HPLC apparatus from Beckman Instruments. Samples (Steglich and Scheffler, 1982). They were maintained were injected onto the system by a SpectraPhysics Sf in Dulbecco’s MEM containing 35 mM NaHCO,, 1.0 P8270 autosampler and passed through a Brownlee mM aminoguanidine, 12 pg/ml penicillin, and 12 pg/ml Labs RP-300 pre-column and onto a Beckman Ultrastreptomycin and supplemented with 10% Serum Plus sphere Ion Pairing 5 pm C,, (250 mm x 4.6 mm) colmedia supplement (Hazleton-Dutchland Inc), 350 pM umn at 36°C. Two buffers were used to form an acetoproline, and 0.5 M putrescine. The cells were grown as nitrile/methanol gradient. Buffer A contained 0.1 M monolayers at 37°C in 10% CO, and 93% humidity. sodium acetate (pH 4.5) and 9.1 mM octane sulfonate. Polyamine uptake experiments. The cells were Buffer B contained 0.2M sodium acetate (pH 4.51, plated in multiwell plates. After the indicated growth 9.1 mM octane sulfonate, 21% acetonitrile, and 9% period, the growth media was aspirated off and a-MEM methanol. There was one gradient step, a change from (without FBS) containing labeled uptake substrate was 30% Buffer B to 100% Buffer B in 35 min. Buffer B was added. The cells were then incubated at 37°C in 10% maintained a t 100%for 17 min. After the gradient was COz and 93% humidity. After the appropriate incuba- completed, the column was returned to initial condition time, the media was aspirated off and the cells tions in 2 min and equilibrated for 10 min before the washed twice with 1ml ice-cold a-MEM (without FBS) next run. The flow rate was 1 mumin. Detection of containing 20 mM unlabeled substrate and then twice polyamines was accomplished using a fluorescence dewith 1ml ice-cold phosphate buffered saline (PBS). The tector after a post column derivitization with owashed cells were then dissolved by sonication in 0.1 N phthalaldehyde/2-mercaptoethanol (Seiler and KnNaOH and aliquots of this solution were used for pro- odgen, 1985). The reagent was delivered a t 1ml/min in tein quantitation and for determination of radioactiv- solution prepared as follows: 0.05% (w/v) o-phthalaldeity. Protein was measured by the dye binding method hyde (Pierce, Fluka) in 0.4 M boric acid (pH 10.4) conof Bradford (1976) using the dye from Bio Rad. Radio- taining 1%methanol, 0.45% 2-mercaptoethanol, and activity was determined using a Beckman LS-3133T 0.03% (w/v) BRIJ. Fluorescence was activated a t scintillation counter after the addition of 10 ml of ACS 345 nm, and fluorescent intensity measured at I1 (Amersham-Searle). Uptake measurements were 255 nm. Fluorescent emissions were recorded and incarried out under conditions in which uptake was tegrated by a SpectraiPhysics SiP4270 integrator. Eluknown to be linear with respect to time. Where uptake tion times were 27 rnin for putrescine, 42 min for sperwas measured over a time course, the rates were de- midine, and 52 min for spermine. Commercially termined from the slopes of graphs of uptake against available polyamine hydrochlorides were used as stantime obtained by a linear regression analysis to obtain dards. the best fit line. Where the effect of DFMO on polyRESULTS amine uptake activity was quantitated, cells were exposed to 5.0 mM DFMO for 48-72 h prior to uptake The response of the polyamine transport system in measurements. Although the uptake measurements CHO cells to excess intracellular polyamine concentrawere made in a-MEM medium, our previous studies tions was examined by incubation in media containing have shown that similar transport rates were obtained polyamines. This resulted in depressed polyamine upwhen uptake was measured in PBS (Byers and Pegg, take activity (Table 1). A similar response has been reported in mouse neuroblastoma cells (Rinehart and 1989). The polyamine content in CHO and CHODC- cells Chen, 1984). Repression of polyamine transport capacwas elevated by incubation with exogenous poly- ity did not require protein synthesis since cyclohexiamines. These cells were washed extensively with PBS mide did not prevent the effect (Table 1).Since expo(37°C)prior to any measurements of radiolabeled poly- sure of CHO cells to cycloheximide for 12 h did not amine uptake. Polyamine levels were reduced in alter the rate of polyamine transport, the proteins inCHODC- cells by thoroughly washing with PBS (37°C) volved do not appear to turn over rapidly (Table 1). prior to harvesting, replating, and growth in media The reversal of the repression of the polyamine lacking polyamines. Although CHOCD- cells were transport system was examined by removing CHO cells grown in Dulbecco’s MEM, polyamine uptake was de- from an exogenous polyamine source (Fig. 1).CHO termined by incubating these cells in a-MEM, without cells maintained in media containing polyamines exFBS, containing radiolabeled substrate. hibited depressed polyamine transport activity. ReSeparation and quantitation of polyamines. The placement of this media with fresh media lacking polyintracellular polyamine content of CHO and CHODC- amines resulted in a time dependent increase in cells was determined using the following procedures. putrescine (Fig. 1A) and spermine (Fig. 1B) uptake.
462
BYERS AND PEGG
TABLE 1. Response of polyamine transport to exogenous polyamines and to cycloheximide'
Treatment None None Cycloheximide, 10 pgiml 50 pM putrescine 5 pM spermine Cycloheximide, 10 pgiml + 50 LLMuutrescine 5 LLMsnermine
+
Transport activity (nmolimg proteidh) Putrescine Spermine 3.3 t 0 . 3 5.3 ? 0.6 4.1 ? 0.2 4.6 ? 0.6 3.0 i 0.2 3.5 t 0.2 0.3 ? 0.2 0.9 i 0.2
Time (h) 0 12 12 12
+
10.3
12
0.5
?
0.4
'CHO cells were plated in the presence of 2 mM aminoguanidine a t 3 x lo4 cells/ml/well. At 72 h putrcscine, spermine, and cyclohcximide were added to the appropriate wells to give 50 p M , 5 pM,and 10 pgiml concentrations, respectively. Determination of putrescine and spermine uptake was carried out a t this time and 12 h later as indicated. The results reported are the means S.D. for 3 observations.
*
1
r
I
.& O2 t l , , a
Hours
g
o
6
,
I
12
,
I
18
,
I
24
Hours
Fig. 1. Effect of removal of an exogenous polyamine source on polyamine uptake in CHO cells. CHO cells were grown in a-MEM containing no additions (*); 2.0 mM aminoguanidine, 50 pM putrescine, and 5 pM spermine (m); or 2.0 mM aminoguanidine, 50 pM putrescine, 5 pM spermine and 5 mM DFMO (A) for 48 h. At the end of this time, the cells were washed and media added back containing no additions
(Control, *), 2.0 mM aminoguanidine (-PA, m) or 2.0 mM aminoguanidine and 5 mM DFMO (-PA+ DFMO, A). After incubation for the indicated periods, the media was removed and putrescine (A) or spermine (B) transport was measured. These values are the means * S.D. for 3 observations.
Transport activity rose to levels slightly above those in cells not exposed to polyamines and then fell to the expected control values. The 6 h peak in control cell polyamine uptake may have been in response to the addition of fresh serum as reported in other cell types (Pohjanpelto, 1976; DiPasquale et al., 1978; Wallace and Keir, 1981). The presence of DFMO did not alter the rate of response to polyamine removal, but did extend and magnify the increase in polyamine transport activity past the peak observed in untreated cells. This increase may be explained by a fall in intracellular polyamines below control levels, due to the inhibition of putrescine synthesis. Protein synthesis was mandatory for the activation of polyamine transport. The addition of cycloheximide a t the time of exogenous polyamine removal prevented the rise in polyamine transport activity observed in cells not exposed to this protein synthesis inhibitor (Fig. 2). The effect of cycloheximide exposure on the activation of polyamine transport was not due to decreased viability or cell death. CHO cells maintained in the presence of polyamines until the addition of cycloheximide had low polyamine uptake activity over a 12 h period. However, removal of this drug a t 12 h resulted in a rise in polyamine uptake equivalent to that
observed in cells not exposed to cycloheximide (data not shown). The inability of CHODC- cells to produce putrescine was exploited to deplete polyamine content without the use of DFMO. Polyamine starvation of CHODC- cells resulted in reduced putrescine and spermidine levels and a n increase in putrescine and spermine uptake which was maintained for up to 72 h and was not amplified by the presence of DFMO. In a typical experiment, growth of CHODC- cells in the absence of polyamines for 72 h resulted in putrescine and spermine uptake values of 7.0 2 0.24 and 5.6 It_ 0.6 nmollmgi protein/h, respectively; cells starved in the presence of DFMO had the respective values of 6.6 k 0.4 and 7.2 +0.6 nmol/mg protein/h. As shown in Figure 3, re-feeding polyamine starved CHODC- cells with exogenous putrescine (Fig. 3A) or spermine (Fig. 3B) resulted in a n immediate increase in the respective intracellular polyamine content and a time dependent decrease in polyamine transport activity. Within 6 h, the transport capacity for either putrescine or spermine was maximally reduced. The total intracellular level of the added polyamine declined rapidly following the repression of transport (Fig. 3). The treatment with putrescine, which elevates intra-
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REGULATION OF POLYAMINE TRANSPORT
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taining 2 mM aminoguanidine (FCyclo, 0 ) or 2.0 mM aminoguanidine and 10 pg/ml cycloheximide ( + Cyclo, m). After incubation for the indicated periods, the media was removed and putrescine (A)or spermine (B) transport was measured. These values are the means f S.D. for 3 observations.
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Fig. 3. Effect of putrescine and spermine exposure on the polyamine transport activity and the polyamine profile of C H O D C cells previously starved for polyamines. CHODC- cells were plated in 35 mm dishes and grown in Dulbecco's MEM in the absence of any polyamine. At 72 h the cells contained putrescine or spermidine levels of C1.5 n m o l h g protein and the spermine content was 9.3 i 1.0 nmoli
mg. At this point, cultures were exposed to 500 p M putrescine (A) or 10 pM spermine (B).After the indicated times, cells were washed 3 X with 5 ml of PBS and harvested for polyamine analysis ( 0 , putrescine; A, spermidine; spermine) or for measurement of transport of putrescine ( 0 )or spermine (0). The values above are the means 2 S.D. for 3 dishes.
cellular spermidine and spermine as well as putrescine, led to an equally rapid decline in putrescine and spermine transport (Fig. 3A). However, exposure to exogenous spermine which increases only spermine seemed to have a somewhat more rapid effect on putrescine transport (Fig. 3B). The addition of a range of putrescine concentrations to polyamine starved CHODC- cells and measurement of the polyamine transport capacity and intracellular polyamine levels 1.5 and 3 h later confirmed that there was a decrease in polyamine transport which was dependent on both time and the accumulation of intracellular polyamines (Table 2). Cells exposed for 1.5 h to extracellular putrescine concentrations above the K, for putrescine uptake, which is about 4.5 pM (Byers et al., 1989), showed elevated polyamine levels and depressed polyamine transport activity. In contrast, CHODC- cells incubated with only 1 pM putrescine had only slightly elevated polyamine contents and did
not change transport activity. However, CHODC- cells exposed to 5 pM putrescine for 1.5 h also exhibited almost control uptake activity despite high intracellular polyamine levels. These results suggest that intracellular polyamine content must reach a threshold after which transport decreases via a time dependent mechanism. Presumably, the polyamine levels in cells exposed to putrescine concentrations allowing for the maximal rate of uptake rapidly reached this threshold level and the effects of these levels on transport are evident by 1.5 h. Cells exposed to 5 pM putrescine may have taken longer to accumulate the threshold level of polyamines, as evidenced by the persistence of transport activity at 1.5 h. However, by 3 h, the transport activity in these cells was also decreased (Table 2). The cells exposed to only 1 pM putrescine showed only a small reduction on transport capacity at 3 h by which time intracellular polyamines had increased substantially (Table 2).
.,
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BYERS AND PEGG
TABLE 2.The effect of incubating cells previously starved for polyamines with various concentrations of exogenous putrescine on Dolvamine transDort activity and intracellular Dolvamine levels' Exogenous putrescine (pM) 0 50 10 5 1 50 10 5 1
Time (h) 0 1.5 1.5 1.5 1.5 3.0 3.0
3.0 3.0
Transport activity (nmolimg protein/h) Putrescine Spermine 7.3 ? 0.6 7.3 2 0.9 1.0 f 0.3 2.7 2 0.3 3.8 f 0.5 2.9 0.4 5.4 0.7 5.0 f 0.9 5.8 ? 0.4 4.7 ? 0.8
