Evaluation method for polyamine uptake by N1-Dansylspermine

Article abstract of DOI:10.1007/s00726-009-0414-9

Polyamine uptake by the polyamine transport system (PTS) in HTC cells was studied without the use of radioisotope-labeled polyamines. N-Dansylspermine (DNS343) was selected as a candidate probe to examine the PTS. DNS343 was incorporated into HTC cells, and its distribution in the cells was confirmed by fluorescence microscopy. The incorporation of DNS343 via PTS was confirmed by a competition study with bis(3-aminopropyl)amine, which is incorporated into cells via the PTS. In addition, the temperature dependency of DNS343 uptake and studies with inhibitors of ornithine decarboxylase and proteoglycan synthesis supported the use of DNS 343 as a fluorescent probe for the PTS. The kinetics studies for HTC cells treated with or without an ornithine decarboxylase inhibitor indicated that DNS343 uptake was saturable and that the apparent Km values for the PTS were approximately 1.5 μM in both types of cells at 37°C. Thus, we developed an assay method for the PTS by high-performance liquid-chromatography with DNS343. The inhibitory effect of polyamine analogs and related compounds on DNS343 uptake was then examined and discussed. Springer-Verlag 2009.

Full text of DOI:10.1007/s00726-009-0414-9

Amino Acids (2010) 38:533–539
DOI 10.1007/s00726-009-0414-9
ORIGINAL ARTICLE
1
Evaluation method for polyamine uptake by N -dansylspermine
Koichi Takao
•
Yoshiaki Sugita Akira Shirahata
•
Received: 27 August 2009 / Accepted: 20 October 2009 / Published online: 3 December 2009
Ó Springer-Verlag 2009
Abstract Polyamine uptake by the polyamine transport
system (PTS) in HTC cells was studied without the use of
Abbreviations
17-DAH
33
1,7-Diaminoheptane
1
radioisotope-labeled polyamines. N -Dansylspermine
Bis(3-aminopropyl)amine
0
(
DNS343) was selected as a candidate probe to examine
333
N,N -Bis(3-aminopropyl)-1,3-
diaminopropane
the PTS. DNS343 was incorporated into HTC cells, and its
distribution in the cells was confirmed by fluorescence
microscopy. The incorporation of DNS343 via PTS was
confirmed by a competition study with bis(3-aminopro-
pyl)amine, which is incorporated into cells via the PTS. In
addition, the temperature dependency of DNS343 uptake
and studies with inhibitors of ornithine decarboxylase and
proteoglycan synthesis supported the use of DNS343 as a
fluorescent probe for the PTS. The kinetics studies for HTC
cells treated with or without an ornithine decarboxylase
inhibitor indicated that DNS343 uptake was saturable and
that the apparent Km values for the PTS were approxi-
mately 1.5 lM in both types of cells at 37°C. Thus, we
developed an assay method for the PTS by high-perfor-
mance liquid-chromatography with DNS343. The inhibi-
tory effect of polyamine analogs and related compounds on
DNS343 uptake was then examined and discussed.
33333
1,19-Diamino-4,8,12,16-
tetraazanonadecane
3334
1,16-Diamino-4,8,12-triazahexadecane
1,20-Diamino-4,8,13,17-tetraazaeicosane
1,17-Diamino-4,9,13-triazaheptadecane
1,21-Diamino-4,9,13,18-
33433
3434
34343
tetraazahenicosane
3443
353
1,17-Diamino-4,9,14-triazaheptadecane
1,13-Diamino-4,10-diazatridecane
1,16-Diamino-4,13-diazahexadecane
1,17-Diamino-5,9,13-triazaheptadecane
1,13-Diamino-5,9-diazatridecane
Homospermidine
383
4334
434
44
444
1,14-Diamino-5,10-diazatetradecane
1,12-Diamino-4-azadidecane
1-Aminooxy-3-aminopropane
Acetylspermine
83
AOAP
Ac343
Keywords Polyamine ꢀ Polyamine transport system ꢀ
Boc O
2
Di-tert-butyl dicarbonate
Diacetylspermine
1
HTC cells ꢀ N -dansylspermine ꢀ HPLC assay ꢀ
DA343
1
17
Polyamine analog
DA3443
N , N -Diacetyl-1,17-diamino-4,9,
4-triazaheptadecane
1
DE33
Diethylnorspermidine
Diethylnorspermine
DE333
DFMO
DNS333
DNS343
DNS-Cl
FBS
a-Difluoromethylornithine
N -Dansylnorspermine
1
K. Takao (&) ꢀ Y. Sugita ꢀ A. Shirahata
Laboratory of Cellular Physiology,
Department of Clinical Dietetics and Human Nutrition,
Faculty of Pharmaceutical Sciences,
Josai University, Keyaki-dai 1-1,
1
N -Dansylspermine
Dansyl chloride
Fetal bovine serum
1
N -Guanidino-1, 7-diaminoheptane
Morris hepatoma 7288c cell
GC7
Sakado, Saitama 350-0295, Japan
e-mail: ktakao@josai.ac.jp
HTC cell
123

5
34
K. Takao et al.
1
4
The PTS activity is measured to screen for agents that
block polyamine transport, and radioisotope-labeled poly-
amines are generally used in this assay (Hoshino et al.
MDL72527
N , N -Bis(2,3-butadienyl)-1,
-butanediamine
4
MGBG
NBD
Methylglyoxalbisguanylhydrazone
1
N -(7-Nitro-2,1,3-benzoxadiazolyl)
1
N -(7-Nitro-2,1,
2005). Although this method is sensitive and convenient,
its use is limited due to radioisotope contamination.
Recently, several groups have shown that fluorescent-
labeled polyamines could be used as probes for the PTS in
flow cytometry analysis (Guminski et al. 2009; Cullis et al.
NBD333
3-
benzoxadiazolyl)norspermine
Ornithine decarboxylase
ODC
Paraquat
PTS
0
0
1,1 -Dimethyl-4,4 -bipyridinium dichloride
Polyamine transport system
1,4-Diaminobutane
1
999; Aziz et al. 1998). In the present study, we developed
1
a sensitive and accurate method that uses N -dansylsper-
mine (DNS343) with a simple HPLC apparatus to evaluate
PTS activity.
Put
Spd
Spermidine
Spm
TCA
TFA
Spermine
Trichloroacetic acid
Trifluoroacetic acid
Materials and methods
Xyl-PheNO2 p-Nitrophenyl-o-b-D-xylopyranoside
Triethylamine, di-tert-butyl dicarbonate (BOC O), trifluo-
2
roacetyl ethylester, dansyl chloride (DNS-Cl), 1,4-Diam-
inobutane (Put) dihydrochloride, bis(3-aminopropyl)amine
Introduction
(norspermidine, 33) and 1,7-diaminoheptane (1,7-DAH)
that were re-crystalized as hydrochloride salts and sperm-
ine (Spm) tetrahydrochloride were purchased from Tokyo
Chemical Industry (Tokyo, Japan). o-Phthaldialdehyde, p-
The cellular content of polyamines, putrescine, spermidine
and spermine, is regulated by biosynthesis, degradation,
and transport. The molecular biology of the biosynthesis,
degradation and interconversion of polyamines has been
well documented, and some properties of the polyamine
transport system (PTS) in mammalian cultured cells have
been determined (Tabor and Tabor 1984; Cohen 1998; I-
garashi and Kashiwagi 2000; Seiler et al. 1996; Belting
et al. 2003; Soulet et al. 2004; Hoshino et al. 2005;
Phanstiel et al. 2007). The PTS is inducible and saturable.
Depletion of the intracellular polyamine content led to a
marked increase in the rate of polyamine uptake that
scarcely affected the affinities to polyamines, while an
increase in the intracellular polyamine level downregulated
the PTS activity. A mechanism-based inhibitor of ornithine
nitrophenyl-o-b-D-xylopyranoside (Xyl-PheNO ), amino-
2
guanidine bicarbonate salt, methylglyoxalbisguanylhy-
0
0
drazone (MGBG) dihydrochloride, 1,1 -dimethyl-4,4 -
bipyridinium (Paraquat) dichloride and agmatine sulfate
were purchased from Sigma (Tokyo, Japan). Trifluoro-
acetic acid (TFA) and 1-octanesulfonic acid sodium salt
were purchased from Wako Pure Chemical Industries
(
Tokyo, Japan). Spermidine (Spd) trihydrochloride and
0
N,N -Bis(3-aminopropyl)-1,3-diaminopropane (333) that
was re-crystalized as hydrochloride salt were purchased
from Aldrich (Tokyo, Japan). Other polyamines, poly-
amine analogs, and fluorescent polyamines were prepared
according to the methods described previously (Takao et al.
decarboxylase
(ODC),
a-(difluoromethyl)ornithine
DFMO) (Mitchell et al. 1992), or a competitive inhibitor
2
007, 2008; Samejima et al. 1984; Niitsu and Samejima
986). All other reagents and organic solvents were of
(
1
of ODC, 1-aminooxy-3-aminopropane (AOAP) (Hyvonen
et al. 1988), caused a substantial depletion of intracellular
putrescine and spermidine that inhibited cell growth and
increased PTS activity. The cellular polyamine content and
cell growth were restored by supplementing exogenous
polyamines to the culture medium (Mamont et al. 1981;
Mitchell et al. 1992). Evidence from several studies sup-
ports the conclusion that the effectiveness of ODC inhibi-
tion could be increased by interfering with the PTS (Ask
et al. 1992; Weeks et al. 2000; Belting et al. 2002).
Extracellular sources of polyamines are important for the
growth of tumor cells; thus, the identification of pharma-
cological agents that block polyamine transport is desired
for the development of antineoplastic drugs (Marton and
Pegg 1995).
commercial analytical grade.
Synthesis of DNS343 tetrahydrochloride
DNS343 was synthesized by a previously described
method (Takao et al. 2008). Briefly, spermine was pro-
tected on one of the primary amines with ethyl trifluoro-
acetate to afford mono-trifluoroacetamide derivative
contaminated with di-trifluoroacetamide derivative. The
remaining free amines were immediately protected with di-
tert-butyl dicarbonate, and then the TFA-protecting group
was removed with ammonia to afford tri-Boc-spermine that
was purified by silica gel chromatography. The tri-Boc-
spermine was fluorescent-labeled with DNS-Cl. The dansyl
1
23

Evaluation method for polyamine transport system
535
derivative was subjected to silica gel column chromatog-
raphy with a solvent system of chloroform:metha-
demonstrated at amounts greater than 20 pmol. The
determination of 33 in Fig. 3 was performed using an ion-
exchange o-phthalaldehyde-post-column HPLC as descri-
bed previously (Shirahata et al. 1993). Aliquots of TCA
supernatants from HTC cells were injected, and 33 was
determined using the peak area.
nol:ammonia to obtain
a single band of tri-Boc-
dansylspermine. Boc groups of the pure derivative were
then de-protected by trifluoroacetic acid to yield DNS343
trifluoroacetate that was exchanged to a hydrochloride
salt. The DNS343 hydrochloride was re-crystalized
from ethanol–water. DNS343 tetrahydrochloride, FAB-
?
MS: 436 [C H N O S] Anal Calcd for C H N O S
2
Treatment with AOAP and Xyl-PheNO2
2
37
5
2
22 37 5 2
4
HCl: C 45.19; H 7.11; N 12.04. Found: C 45.19; H 6.98;
N 11.97.
Inhibitor treatments were performed according to the pre-
viously described methods (Takao et al. 2007; Belting et al.
5
999). Briefly, cells were seeded at 5 9 10 cells per 10-
1
Cell culture
cm diameter culture dish in the absence or presence of
1
00 lM AOAP, and 1 mM of Xyl-PheNO (final concen-
2
Morris hepatoma 7288c (HTC) cells and MEM-Eagle’s
medium were purchased from Dainippon Pharmaceuticals
tration) was added to the culture 1 day after the seeding.
After 2 days of culture with AOAP, cells were used for the
DNS343 uptake experiment described above.
(
Tokyo, Japan), and fetal bovine serum (FBS) was pur-
chased from Nippon Biotest Laboratories, Inc., (Tokyo,
Japan). Cells were cultured according to previously
described methods (Beppu et al. 1996). HTC cells were
grown as a monolayer in culture dishes. The HTC cells
were maintained in MEM-Eagle medium containing 10%
Fluorescence microscopy
Cells were seeded on a Lab-Tek2 chamber slide system
(Nalge Nunc International, Tokyo, Japan). The cells
attached to the slide were treated with 10 lM DNS343 for
FBS at 37°C in an atmosphere of 5% CO and 95% air.
2
6
h and fixed by formaldehyde in the usual manner. The
DNS343 uptake experiment
fixed cells were viewed with an Olympus BX51WI upright
microscope. We used a specific filter for DNS343 (Omega
Optical, Ex/Em = 330/535 nm) to observe the fluores-
cence of compounds containing the dansyl group.
5
Cells were seeded at 5 9 10 cells per 10-cm diameter
culture dish. Cells grown for 24 h after the seeding were
used for the experiments. The medium containing 1 mM
aminoguanidine as an inhibitor of diamine oxidase and
1
0 lM DNS343 (10 ml) was added to the monolayer.
Results and discussion
Various compounds to be tested were added as described in
the text. After incubation for 2 h at 37°C, the attached cells
were washed three times with PBS, scraped, harvested,
counted, and centrifuged by the usual method. A total of
To identify a suitable fluorescent probe for the PTS, dansyl
1
derivatives and N -(7-nitro-2,1,3-benzoxadiazolyl) (NBD)
derivatives of polyamines were examined instead of a large
polyaromatic fluorophore, which might non-specifically
interact with the lipid bilayer. Three compounds, DNS343,
2
00 ll of 10% trichloroacetic acid (TCA) was added to the
resulting cell pellets; then, the mixture was vortexed and
centrifuged. The supernatants were collected and subjected
to HPLC analysis.
1
1
N -dansylnorspermine (DNS333), and N -NBD norsper-
mine (NBD333), were prepared, and their incorporation
into HTC cells, which have well-established PTS proper-
ties, was compared (Mamont et al. 1981; Mitchell et al.
1992). The results showed that the DNS343 and DNS333
uptakes were similar and greater than the NBD333 uptake
(data not shown). Thus, DNS343, which has the natural
polyamine (spermidine) structure, was used for the fol-
lowing study.
HPLC analysis
HPLC conditions for DNS343 were as follows: column,
Tosoh ODS-80T (4.6 mm/ 9 150 mm); isocratic elution
M
solution, methanol:water (75:25) containing 8 mM sodium
octanesulfonate and 0.1% trifluoroacetic acid; flow rate
HPLC chromatograms of authentic DNS343 and sample
solutions from HTC cells incubated in the presence or
absence of DNS343 are shown in Fig. 1. DNS343 incor-
porated into HTC cells was clearly detected as a single
peak.
1
ml/min; and fluorescence detection by a Shimadzu fluo-
rescence detector RF-10AXL, Ex: 333 nm and Em 544 nm.
The detection limit of DNS343 was approximately
3
0 fmol, and a linearity of the peak area of DNS343 was
123

5
36
K. Takao et al.
Standard
DNS343 (30 pmol)
Non-treated cells
Treated cells
DNS343
(
% )
00
Uptake of 33
Uptake of DNS343
1
8
0
0
0
0
6
4
2
0
0
µM
100
µM
0
µM
100 µM
DNS343
33
Fig. 3 Competition study of the uptake of 33 or DNS343 into HTC
cells. Cells were incubated in the presence of 10 lM 33 alone or plus
100 lM DNS343 and in the presence of 10 lM DNS343 alone or plus
(
min)
(min)
(min)
0
5 10
0
5 10
0 5 10
1
00 lM 33. Data are expressed as means ± SD (n = 3)
Fig. 1 HPLC chromatograms of authentic DNS343 and DNS343-
treated or non-treated cells. HPLC conditions are described in the
Materials and methods. Authentic DNS343 (30 pmol) was injected
3
2
2
1
1
000
500
000
500
000
(
standard). HTC cells were incubated in the absence (non-treated
cells) or presence (treated cells) of 10 lM DNS343 for 2 h, and then
samples for injection to HPLC were prepared
500
0
0
1
2
3
4
Incubation time ( hr )
Fig. 4 Time-dependent uptake of DNS343 into HTC cells under
different conditions. Cells were pre-cultured in the absence (control)
or presence of 100 lM AOAP for 2 days and subjected to the method
for the measurement of DNS343 uptake. Cell samples were collected
at the indicated culture time. Control cells were cultured at 37°C
(
filled triangle) or 0°C (open circle), and AOAP-treated cells were
cultured at 37°C (open square). Data are expressed as duplicate
determinations
Fig. 2 Fluorescence photomicrograph of DNS343 accumulation in
HTC cells seeded on a sterile glass chamber slide. Cells were grown
on the Lab-Tek2 chamber slide system. After attachment, cells were
incubated in the presence of 10 lM DNS343 and fixed with
formaldehyde. The fluorescence of the dansyl group was detected
by an Olympus BX51WI upright fluorescence microscope with filter
for DNS343 (Omega Optical, Ex/Em = 330/535 nm)
(
33), which is incorporated into cells via the PTS as a
spermidine analog (Porter 1983). The presence of higher
concentrations of 33 in the media inhibited the cellular
uptake of DNS343, and also the uptake of 33 was inhibited
by DNS343 (Fig. 3). These results suggested that DNS343
was incorporated via the PTS.
DNS343 was incorporated into HTC cells in a linear
manner for more than 4 h (Fig. 4). Although Seiler et al.
reported that DNS343 was a potent calmodulin antagonist
that interfered with the PTS (Seiler et al. 1998), the linear
DNS343 uptake strongly suggested that DNS343 does not
have any effect on the PTS as a calmodulin antagonist in
our conditions. In addition, the uptake was temperature-
dependent; only negligible uptake occurred at 0°C, as
compared to the significant uptake at 37°C in control cul-
tures. The incorporation of DNS343 into HTC cells treated
As shown in the fluorescence photomicrograph of
DNS343-treated cells (Fig. 2), the fluorescence was mainly
located in the area of cytoplasm. This finding was similar
to the results of previous studies that used N-(4,4-difluoro-
5
,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-
0
4
N -{S-[spermidine-(N -ethyl)]thioacetyl}ethylenediamine
(
Kaneko et al. 2007; Soulet et al. 2002).
To assess the uptake of DNS343 via the PTS, a compe-
tition study was performed with bis(3-aminopropyl)amine
1
23

Evaluation method for polyamine transport system
537
Table 1 Inhibition of DNS343 uptake into HTC cells by polyamines
or related compounds
2
1
000
000
0
Polyamines or
related compounds
DNS343 uptake (percentage of control)
1
lM
10 lM
100 lM
Diamines
MDL72527
AOAP
[95
[95
[95
70
[95
[95
[95
42
[95
[95
82
0
10
20
30
40
50
Put
Concentrations of DNS343 ( µM )
1
,7-DAH
10
Fig. 5 Dose-dependent uptake of DNS343 into HTC cells. Cells
were pre-cultured in the absence (control filled triangle) or presence
of 100 lM AOAP (open square) for 2 days and subjected to the
method for the measurement of DNS343 uptake. Cells were cultured
with the indicated concentration (0.5–50 lM) of DNS343 for 2 h.
Data are expressed as means ± SD (n = 3)
Triamines
DE33
[95
90
[95
60
32
16
9
74
11
9
8
3
33
81
Spd
71
\5
\5
4
4
55
with AOAP, which prevents putrescine production at a
concentration lower than DFMO (Hyvonen et al. 1988;
Poulin et al. 1989; Takao et al. 2007) and increases the PTS
activity (Hyvonen et al. 1988), also increased in a linear
manner for more than 4 h.
Tetramines
DA343
[95
[95
75
[95
90
60
60
28
11
9
95
41
Ac343
3
83
23
DE333
86
13
A tentative kinetics study for HTC cells treated with or
without AOAP indicated that DNS343 uptake was satura-
ble and that the apparent Km values for the PTS were
approximately 1.5 lM in both types of cells at 37°C
3
4
4
3
33
34
44
53
71
11
32
\5
\5
\5
\5
30
32
6
(
Fig. 5). This Km value is similar to a previously reported
Spm
29
6
1
4
Km value measured with C-labeled spermidine in HTC
cells (Mitchell et al. 1992).
Pentamines
DA3443
93
56
22
20
14
76
12
6
48
6
Bolkenius and Seiler (1981) reported in vitro degrada-
tion of DNS343 by polyamine oxidase (PAO). Under the
present incubation conditions for 2 h, there was no differ-
ence in the incorporation of DNS343 with or without
3334
3434
4334
3443
\5
\5
\5
\5
\5
2
5 lM MDL72527, an irreversible inhibitor of PAO (data
Hexamines
not shown). A PAO inhibitor, such as MDL72527, which
does not have any effect on DNS343 uptake (Table 1),
should be added to the incubation media in uptake exper-
iments when DNS343 is applied to cells with high PAO
activity, such as Chinese hamster ovary cells (Carper et al.
33333
33433
34343
35
31
20
8
\5
\5
\5
8
\5
Others
MGBG
Paraquat
GC7
[95
[95
84
95
94
73
79
81
78
38
31
1
991).
Belting et al. (1999) reported that proteoglycans were
involved in polyamine uptake; this conclusion was based
on their observation that spermine uptake into HEL cells
Agmatine
91
was inhibited by Xyl-PheNO , an inhibitor of endogenous
2
Cells were incubated for 2 h in the presence of 10 lM DNS343 and 1,
10, or 100 lM polyamines or related compounds. The number of
control cells and treated cells remained equivalent during the exper-
iments. Data are expressed as means (n = 3)
proteoglycan synthesis. The effect of Xyl-PheNO2 on
DNS343 uptake was then examined. The uptake decreased
with Xyl-PheNO2 and increased with AOAP (Fig. 6),
assuring the use of DNS343 as a probe for the PTS.
Based on these observations, DNS343 was applied to
examine the affinities of various polyamines and related
compounds to the PTS. The inhibition of DNS343 uptake
with these compounds is summarized in Table 1.
DA343 including Put, DE33, MGBG and Paraquat, might
not necessarily enter cells via the PTS. Acetylation of the
terminal amine group of polyamines significantly reduced
their inhibitory effects on DNS343 uptake, as illustrated by
the data from DA343, Ac343 and Spm and from DA3443
and 3443. Compounds with a terminal primary amine were
Compounds that showed no inhibition or weak inhibi-
tion of DNS343 uptake, such as MDL72527, AOAP and
123

5
38
K. Takao et al.
3
2
2
1
1
00
50
00
50
00
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cells. Cells were seeded in the absence (control) or presence of
2
on the uptake of DNS343 into HTC
1
00 lM AOAP. The next day, 1 mM Xyl-PheNO
2
was added. After
1 day of incubation, the DNS343 uptake of cells was assayed. Data
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inhibition. Compounds of similar molecular size with a
different charge provided similar levels of inhibition in the
case of 1,7-DAH and 33, but such compounds provided
different levels of inhibition in the case of 83 and Spm and
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Acknowledgments We express our gratitude to Dr. K. Samejima
for his help in preparing the manuscript and to Mr. T. Usui and Dr. Y.
Hibino for their help with fluorescence microscopy.
Niitsu M, Samejima K (1986) Synthesis of a series of linear
pentaamines with three and four methylene chain intervals.
Chem Pharm Bull 34:1032–1038
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