(Received for publication, August 29, 1994; and in revised form, November 14, 1994)
From the
The properties and regulation of the mammalian polyamine
transport system are still poorly understood. In estrogen-responsive
ZR-75-1 human breast cancer cells, which display low polyamine
biosynthetic activity, putrescine and spermidine were internalized with
high affinity (K = 3.7 and 0.5
µM, respectively) via a single class of saturable
transporter shared by both substrate types, or via distinct but closely
similar carriers. The V
, but not the K
of polyamine transport was rapidly and
synergistically up-regulated by estrogens and insulin. The steady decay
in transport activity observed in hormone-deprived cells was
accelerated by retinoic acid. The enhancement of uptake activity
resulting from polyamine depletion was amplified 3-fold by estrogens
and insulin despite profound growth inhibition, indicating that the
cooperative hormonal induction of polyamine transport is dissociated
from cell growth status. Polyamine uptake was under feedback inhibition
by at least three distinct mechanisms in these cells, namely (i) the
induction of a short-lived protein not actively synthesized without
ongoing uptake or upon polyamine deletion; (ii) a more latent, protein
synthesis-independent ``trans-inhibition'' mechanism; and
(iii) a post-carrier, cycloheximide-sensitive mechanism limiting
substrate accumulation. The complexity of these multiple levels of
feedback transport inhibition is in keeping with the cytotoxicity of
excessive polyamine content.
The polyamines spermidine and spermine, and their diamine precursor, putrescine, are essential constituents of all eukaryotic cells(1, 2, 3) . These polycationic compounds are required for growth-related processes, such as the transcription of specific genes (4) , the unique hypusine modification of the eukaryotic translation initiation factor eIF-5A(5) , and other aspects of macromolecular biosynthesis(6) . Although the enzymatic machinery required for polyamine biosynthesis is expressed in the vast majority of nucleated cells(1, 2, 3) , a high affinity polyamine transport system has also been detected in most, if not all cell types examined(7) .
There is substantial evidence that uptake from
extracellular sources plays a quantitatively important role in the
homeostasis of polyamine pools in vivo. For instance,
tumor-forming ability of L1210 mouse leukemia mutant cells defective in
polyamine transport is strongly impaired in hosts treated with
-difluoromethylornithine (DFMO), (
)a suicide inhibitor
of ornithine decarboxylase, unlike parental
cells(8, 9) . The main source of circulating
polyamines is thought to be the gastrointestinal tract, where the
microbial flora generates millimolar concentrations of putrescine and
cadaverine which are made available to the plasma compartment through
enterohepatic circulation(10, 11, 12) .
Moreover, DFMO does not inhibit bacterial pathways of putrescine and
cadaverine biosynthesis(13) . The potential therapeutic
implications of plasma-borne polyamines are made clear by the
demonstration that several invasive tumor types grow very poorly in
DFMO-treated rodents fed with antibiotics and a polyamine-deficient
diet, whereas treatment with DFMO alone has little, if any, antitumor
activity in such models (9, 14) . Cell transformation
is in fact characterized by a strikingly enhanced capacity for diamine
and polyamine uptake(7) . This is reflected in vivo by
a strongly preferential accumulation of radiolabeled polyamines in
tumors relative to healthy
tissues(10, 15, 16) . Thus, the clinical
usefulness of polyamine antimetabolites like DFMO may ultimately be
limited by the capacity of tumors to salvage extracellular polyamines.
However, the kinetic and physiological properties of the mammalian polyamine uptake system are poorly understood, and its structure is as yet completely unknown. There is no consensus on the number of different carrier species involved in the specific uptake of polyamines, although at least one high affinity, saturable transport system appears to be shared by putrescine, spermidine, and spermine in several mammalian cell types(7) . One consistently observed property of polyamine uptake is its negative regulation by intracellular polyamine levels(7) . This is first reflected by the up-regulation of polyamine uptake activity which follows the depletion of intracellular polyamine pools by enzyme inhibitors such as DFMO(2, 7, 17) , and which likely contributes to the resistance of many tumors to growth inhibition by these agents in vivo(8, 9, 14) . Conversely, the rate of polyamine uptake quickly decays following preincubation with exogenous polyamines(17, 18, 19, 20) . The mechanism responsible for the feedback inhibition of polyamine transport is incompletely understood. An apparent relief of basal and/or spermidine-induced inhibition of polyamine uptake by protein synthesis inhibitors has been noted earlier in mammalian cells (21, 22) and in the fungus Neurospora crassa(23) , suggesting a requirement for the synthesis of a short-lived polyamine transport repressor (PTR) protein(22, 23) . Recent evidence strongly suggests that the latter repressor might be the ornithine decarboxylase antizyme or a closely related protein(24, 25) .
Polyamines
play major physiological functions in the mouse mammary gland (21, 26) as well as in human breast
tumors(27, 28, 29, 30, 31) .
Breast tumors have elevated polyamine levels as compared with healthy
tissues (32, 33) and are exquisitely sensitive to
growth inhibition by DFMO in
vitro(27, 28, 29, 30, 31) . ()Estradiol [E
] has been reported to
stimulate ornithine decarboxylase expression in estrogen-responsive
human breast cancer cell lines(27, 29, 34) ,
but more recent evidence suggests that estrogens act mostly to
cooperatively enhance the induction of ornithine decarboxylase by
polypeptide growth factors such as insulin and insulin-like growth
factor-1. (
)Earlier studies have demonstrated that polyamine
uptake is developmentally regulated and under control by insulin and
prolactin in the normal mouse mammary gland(21) . Although the
hormonal regulation of polyamine biosynthesis in human breast cancer
cells has been the focus of several
studies(27, 28, 29, 30, 34) ,
the polyamine transport system has not been previously characterized in
these tumors. The fact that ornithine decarboxylase expression is
characteristically low in many breast epithelial carcinoma cells
(27) suggests that these tumors might actively rely on
uptake of exogenous polyamines, or on an efficient system for salvaging
polyamines spontaneously released (35, 36) in the
tumor microenvironment.
We are now reporting the kinetic characteristics, substrate specificity, as well as the hormonal and feedback regulation of the polyamine transport system in estrogen-responsive human breast cancer cells. These cells are shown to possess a very active and specific polyamine uptake system with quite narrow structural constraints for substrate recognition. Moreover, estrogens and insulin are not only potent, cooperative inducers of polyamine transport, but are also required for its up-regulation by polyamine depletion in the absence of cell growth. Finally, evidence is presented that net polyamine accumulation is restricted not only by an unstable PTR, but also by other feedback inhibition mechanisms acting on carrier activity as well as at a post-transporter level. The physicochemical requirements of this transport system are described in the companion article(37) .
N-(3-Aminopropyl)hexahydropyrimidine (APHHP; cf.Table 1) was synthesized in 70% yield by treating sym-norspermidine (N,N`-{bis(aminopropyl)}-1,3-diaminopropane)
with a 37% (w/w) aqueous solution of formaldehyde in tetrahydrofuran at
room temperature(40) ;
H NMR (in CDCl
):
1.33 (bs, 2 H, NH
), 1.53-1.63 (m, 4 H,
CH
), 2.26 (t, 2 H, CH
N, J = 7.5
Hz), 2.53 (bs, 1 H, NH), 2.70 (t, 4H, CH
N, J = 6.7 Hz), 2.78 (t, 2 H, CH
N, J = 5.7 Hz), 3.34 (bs, 2H, NCH
N);
C
NMR (CDCl
):
69.71, 52.90, 52.82, 44.86, 40.42, 30.27,
26.80. 3-Cyclohexylpropylamine (cf. Table 1) was
synthesized as follows. Briefly, a mixture of 3-cyclohexylpropyl
chloride (16 g, 100 mmol) and potassium phthalimide (18.5 g, 100 mmol)
in dimethylformamide (200 ml) was refluxed for 3 h, cooled to room
temperature to give N-3-cyclohexylpropyl phthalimide (25.3 g,
93%)
H NMR (CDCl
):
0.83-0.86 (m, 2
H), 108-1.34 (m, 6 H), 1.45-1.8 (m, 7 H), 3.65 (t, 2H, J = 7.3 Hz), 7.68-7.77 (m, 2H), 7.77-7.87
(M, 2H);
C NMR (CDCl
):
168.42, 133.79,
132.16, 123.10, 38.33, 37.30, 34.41, 33.21, 26.59, 26.30, 25.97. The
crude phthalimide was treated with 5 ml of hydrazine monohydrate in 100
ml of ethanol and refluxed for 40 min. The mixture was cooled to room
temperature, filtered, washed with CHCl
to give the crude
product which, following in vacuo distillation, yielded 1 g of
the pure 3-cyclohexylpropylamine (6.4% yield);
H NMR
(CDCl
):
0.84-0.96 (m, 2 H), 105-1.27 (m,
6 H), 1.39-1.75 (m, 9 H), 2.65 (t, 2H, J = 6.9
Hz);
C NMR (CDCl
):
42.36, 37.33, 34.44,
33.18, 30.87, 26.46, 26.15.
For the determination of
kinetic parameters of transport, the substrate concentration was varied
by adding increasing concentrations of nonradioactive substrate to a
fixed amount of [H]putrescine and
[
H]spermidine, and the K
and V
values were determined by Lineweaver-Burk
analysis. Competition for uptake by various compounds was measured by
adding increasing concentrations of the test substance in the presence
of a fixed amount of radioactive substrate (3 µM [
H]putrescine or 1 µM [
H]spermidine, respectively). The effective
concentration of competitor needed for half-maximal uptake inhibition
(IC
) was obtained by iterative curve fitting of a
sigmoidal equation using the SigmaPlot software program (Jandel
Scientific, Corte Madera, CA), and K
values were
calculated using Dixon plots(43) .
Figure 1:
Kinetic analysis of
putrescine and spermidine uptake in ZR-75-1 human breast cancer
cells. [H]Putrescine (
) and
[
H]spermidine uptake (
) was determined as
described under ``Experimental Procedures.'' Each point is
the mean ± S.D. of determinations from triplicate cultures. Insets, Lineweaver-Burk plots of the
results.
Figure 2:
Inhibition of putrescine and spermidine
uptake by common substrates of the transport system in ZR-75-1
cells. [H]Putrescine (A) and
[
H]spermidine uptake (B) were determined
using 10 and 1 µM of substrate, respectively, in the
presence of increasing concentrations of spermidine (A,
), spermine (
), or putrescine (B,
). Each
point is the mean ± S.D. of determinations from triplicate
cultures. K
values were determined using
Dixon plots(41) .
Figure 3:
Time course of the effect of E and insulin on cell proliferation and spermidine uptake in
ZR-75-1 cells. ZR-75-1 cells were grown for the indicated
period in RPMI 1640 medium supplemented with 5% (v/v) dextran-coated
charcoal-treated fetal bovine serum containing either no hormone
addition (
), 10 µg/ml of insulin (
), 1 nM E
(
), or the combination thereof (
), and
analyzed for DNA content (A) and spermidine uptake activity (B). Each point is the mean ± S.D. of determinations
from triplicate cultures. When no bar is shown, the experimental
deviation was smaller than the symbol used.
Figure 4:
Effect of DFMO, insulin and E on growth and putrescine uptake in ZR-75-1 cells. A and B, ZR-75-1 cells were grown for the indicated
time in RPMI 1640 medium supplemented with 5% (v/v) dextran-coated
charcoal-treated fetal bovine serum containing either no hormone
addition (control,
), 500 µM DFMO (
), 10
µg/ml insulin and 1 nM E
(
), or both
DFMO and the hormone combination (
), and assayed for DNA content (A) and putrescine uptake (B). C,
Lineweaver-Burk analysis of putrescine uptake characteristics in
ZR-75-1 cells treated for 4 days as in B.
Mitogenic stimulation has been
temporally associated with increased polyamine transport in other cell
types(19, 46, 47) . On the other hand,
depletion of intracellular putrescine and spermidine by DFMO also
increases the rate of polyamine uptake, presumably by relieving
endogenous feedback inhibition of
transport(2, 7, 17) . Since DFMO potently
inhibits breast cancer cell proliferation(27, 28, 29, 34) , the
relationship between the enhancement of polyamine transport by E
and insulin and their mitogenic effect was further examined by
comparing the effect of DFMO on putrescine uptake activity in the
presence or absence of these hormones. Preliminary experiments had
shown that the hormone ablation-induced decay in putrescine uptake
activity could be accelerated by increasing the plating density (data
not shown). Thus, this stepdown strategy was used here to better
evaluate the up-regulatory effect of hormone treatment on putrescine
uptake activity. DFMO had a limited inhibitory action on the already
low growth rate observed in the absence of mitogens (Fig. 4A), and stably increased the rate of putrescine
transport by about 33% as early as 24 h after its addition (Fig. 4B). On the other hand, DFMO almost completely
blocked the mitogenic effect of insulin and E
, while
potentiating the associated increase in putrescine uptake activity. As
shown in Fig. 4C, elevations in putrescine transport
activity induced by insulin and E
as well as by DFMO after
a 4-day treatment solely resulted from an increase in the V
of the uptake process. Likewise, the hormonal
and DFMO-induced stimulation of spermidine uptake did not modify
substrate affinity (data not shown).
Figure 5:
Effect of retinoic acid on cell growth and
spermidine transport in ZR-75-1 cells. Three days after seeding
in RPMI 1640 medium supplemented with 5% (v/v) dextran-coated
charcoal-treated fetal bovine serum, cells were preincubated for 2 days
in the presence (,
) or absence (
,
) of
insulin (10 µg/ml) and E
(1 nM). Retinoic acid (RA, 1 µM) (solidsymbols) or
the ethanol vehicle (0.1%, v/v) (plainsymbols) was
then further added at time 0 of the time course shown, and cells were
analyzed for DNA content (A) and spermidine uptake activity (B). Each point is the mean ± S.D. of determinations
from triplicate cultures.
In order to first address the requirement for protein synthesis for feedback transport inhibition in ZR-75-1 cells, the acute effect of spermidine or putrescine on polyamine uptake activity was determined in the presence or absence of cycloheximide (CHX) (Fig. 6A). Exogenous spermidine (20 µM) caused a nearly maximal down-regulation (90% inhibition) of its own uptake rate within 2 h, with an approximately 50% reduction already observed after 1 h, thus indicating the presence of a rapid and efficient mechanism of feedback transport repression in these cells. An equimolar concentration of putrescine was much less potent in that respect, slowly decreasing the velocity of spermidine uptake by about 60% after 6 h. While CHX alone slightly inhibited spermidine uptake activity, it delayed the onset of feedback transport inhibition so that a comparable degree of polyamine-induced repression required about 6 h in its presence. CHX blocked the down-regulation of polyamine transport induced by putrescine much more potently than that caused by spermidine (Fig. 6A).
Figure 6:
Dependence on protein synthesis and
structural specificity of feedback inhibition of spermidine transport
in ZR-75-1 cells. A, at time 0, 20 µM spermidine (,
), 20 µM putrescine
(
,
), or no amine supplement (
,
) was added to
ZR-75-1 cell cultures in standard growth medium containing 1
mM aminoguanidine, in the presence (solidsymbols) or absence (plainsymbols) of
200 µM CHX. [
H]Spermidine uptake
activity was measured at the indicated times after rinsing cell
monolayers twice with serum- and amine-free RPMI 1640 medium. B, prior to the experiment, cells were grown for 6 days in standard
growth medium containing 1 mM aminoguanidine, with 10
µM AbeAdo being added to one group of cells for the last
24 h. Medium was then supplemented with the indicated concentrations of
spermine (
), spermidine (
), putrescine (
), or
putrescine + 10 µM AbeAdo (
). After a 3-h
incubation period, cell monolayers were rinsed twice with serum- and
amine-free RPMI 1640 medium, and [
H]spermidine
uptake activity then determined. Each point is the mean ± S.D.
of determinations from triplicate cultures.
The ability of various substrates to
down-regulate polyamine transport was compared by preincubating
ZR-75-1 cells for 3 h with putrescine (0.3-30
µM), spermidine (0.03-10 µM), or
spermine (0.01-3 µM) prior to the determination of
uptake activity (Fig. 6B). Spermidine and spermine were
both very potent feedback repressors of polyamine transport, with
IC values of 0.09 ± 0.03 and 0.21 ± 0.07
µM, respectively, while putrescine was 25-60-fold
less potent (IC
= 5.4 ± 1.2
µM). Furthermore, pretreating the cells with 10 µM AbeAdo, an irreversible inhibitor of S-adenosylmethionine
decarboxylase (39) which depletes spermidine and spermine while
causing substantial putrescine accumulation(52, 53) ,
did not notably decrease the effective concentration of exogenous
putrescine needed to repress spermidine uptake (IC
3.6 ± 0.7 µM; Fig. 6B). The latter
observation, and the fact that AbeAdo treatment alone increased the
basal rate of spermidine uptake by 40-50%, strongly suggest that
spermidine and spermine are the major effectors for the feedback
inhibition of polyamine transport.
The effect of CHX on the time
course of internalization of H-labeled spermidine (5
µM) in ZR-75-1 cells is shown in Fig. 7.
[
H]Spermidine accumulation reached a maximal,
steady-state level within 2 h, i.e. the time required for a
maximal, protein synthesis-dependent decrease in the uptake rate (cf.Fig. 6A). CHX prevented this
stabilization and led to a linear substrate accumulation (up to
4-fold), which abruptly stopped between 4 and 6 h, in keeping with the
time course observed for the protein synthesis-independent component of
feedback transport inhibition. In order to assess whether maintenance
of the feedback suppression of polyamine uptake required ongoing
protein synthesis, the effect of CHX on
[
H]spermidine accumulation was also monitored in
parallel cell cultures preincubated with 10 µM 1-methylspermidine (MeSpd) for the last 3 h before addition of the
labeled precursor. While MeSpd can functionally replace spermidine in
mammalian cells, it is a very poor substrate for spermine synthase and
is completely resistant to acetylation by spermidine/spermine N
-acetyltransferase(38, 57) , an
enzyme involved in polyamine catabolism and excretion(80) .
This spermidine homolog was thus expected to exert its intracellular
effects longer than the natural polyamines. As shown in Fig. 7,
the rate of [
H]spermidine accumulation was
decreased by about 70% in cells preincubated with MeSpd as a result of
feedback transport inhibition. However,
[
H]spermidine accumulation in MeSpd-treated cells
was increased 3-5-fold by CHX, a significant stimulation being
noted as early as after 30 min, and stopped between 4 and 6 h when
intracellular [
H]spermidine levels had reached
approximately 120 nmol/mg DNA. When taking into account the initial
MeSpd pool already internalized during the preincubation period (cf.Fig. 8A), the total polyamine
(MeSpd + spermidine) accumulation at which net
[
H]spermidine uptake ceased in these cells was
comparable to that measured in CHX-treated cells incubated with
spermidine only (i.e. about 200 nmol/mg DNA).
Figure 7:
Effect of CHX on the time course of
[H]spermidine accumulation in ZR-75-1
cells. ZR-75-1 cells were preincubated for 3 h in the absence
(
,
) or presence (
,
) of 10 µM MeSpd in standard growth medium containing 1 mM aminoguanidine. At time 0, medium was substituted with serum-free
RPMI 1640 medium containing 5 µM [
H]spermidine plus (solidsymbols) or minus (plainsymbols) 200
µM CHX, and intracellular radioactive content was
determined at the intervals shown. C, control cells. Each
point is the mean ± S.D. of determinations from triplicate
cultures.
Figure 8:
Effect of CHX on MeSpd accumulation in
ZR-75-1 cells. Cells were incubated in standard growth medium
containing 10 µM MeSpd in the presence () or absence
(
) of 200 µM CHX and harvested at the times shown for
determination of MeSpd content by high performance liquid
chromatography (A), and total trichloroacetic acid-insoluble
protein content (B). Each point is the mean ± S.D. of
determinations from triplicate cultures.
As for
[H]spermidine, MeSpd accumulation rapidly reached
a maximum within 2 h, thus indicating that MeSpd compares to spermidine
as a feedback transport inhibitor (Fig. 8A). On the
other hand, CHX caused an almost linear rate of MeSpd internalization
(
1 nmol/h/culture) for up to 8 h, resulting in a 4-fold higher
accumulation of the analog. The maximal, net MeSpd accumulation thus
reached (equivalent to 180 nmol/mg DNA) was similar to that observed
with [
H]spermidine after only 4 h (cf.Fig. 7). This buildup was followed by an abrupt decrease in
total MeSpd content per culture (Fig. 8A) coinciding
with an acceleration of net protein loss detected in CHX-treated cells (Fig. 8B). No significant effect on total cellular
protein content was observed in cells incubated for up to 12 h with CHX
in the absence of exogenous polyamines (data not shown), strongly
suggesting that the observed toxicity was related to the accumulation
of the polyamine rather than to the effect of CHX per se.
Figure 9:
Effect of DFMO on feedback inhibition of
spermidine uptake activity. Cells were grown in standard growth medium
for 5 days, with 1 mM DFMO being added to half of the cell
cultures during the last 3 days. Medium was then replaced with
serum-free RPMI 1640 medium plus (B) or minus (A) 1
mM DFMO, and containing either 20 µM spermidine
(,
) or no amine supplement (
,
), in the
absence (plainsymbols) or presence (solidsymbols) of 200 µM CHX, and
[
H]spermidine uptake activity was assayed at the
indicated times. Each point is the mean ± S.D. of determinations
from triplicate cultures.
In order to
compare the effect of DFMO and CHX on intracellular polyamine
accumulation, ZR-75-1 cells were preincubated for 4 days in the
presence or absence of 1 mM DFMO, and the differential effect
of CHX on putrescine and spermidine internalization then determined (Table 3). The main change in polyamine content induced by DFMO
in ZR-75-1 cells was a 20% decrease in the level of spermine, the
major polyamine species in this cell line, while the already low
putrescine and spermidine contents fell below detection limits. Despite
this apparently modest decrease in total polyamine content, DFMO
pretreatment alone increased the net accumulation of exogenous
putrescine and spermidine by 77 and 94%, respectively. While CHX
increased net putrescine and spermidine internalization 2.0-
and 6.8-fold, respectively, its differential effect on putrescine
accumulation was much weaker (+30%, p < 0.05) in the
presence of DFMO. On the other hand, spermidine accumulation was still
markedly sensitive to CHX (3.2-fold increase) in DFMO-treated cells.
Interestingly, putrescine accumulation led to an increase in spermidine
content in both control and DFMO-treated cells, even in the presence of
CHX. This observation is consistent with the low ornithine
decarboxylase expression found in ZR-75-1 cells being
a major limiting factor for polyamine biosynthesis in this cell line.
Low amounts of N
-acetylspermidine (1.0 ±
0.1 nmol/mg protein) were also formed upon spermidine addition in
DFMO-treated, but not in control or CHX-treated cells (data not shown).
As shown in Fig. 10, although DFMO markedly increased the
initial velocity of [
H]spermidine
internalization, feedback inhibition of polyamine accumulation followed
a similar time course in control and DFMO-treated cells, with a virtual
cessation in net internalization observed after about 2 h. Likewise,
DFMO did not qualitatively alter the pattern of
[
H]spermidine internalization observed upon
derepression of transport by CHX, except that the rate of accumulation
was increased 1.5-2-fold.
Figure 10:
Effect of DFMO and CHX on
[H]spermidine accumulation in ZR-75-1
cells. Prior to the experiment, cells were grown in the presence or
absence of 1 mM DFMO as described in Fig. 9. At time 0,
serum-free medium containing 5 µM [
H]spermidine was added in the absence (plainsymbols) or presence (solidsymbols) of 200 µM CHX to control (
,
) or DFMO-treated cells (
,
). Intracellular
radioactive content was determined at the indicated times. The
ornithine decarboxylase inhibitor was present throughout the
experimental period. Each point is the mean ± S.D. of
determinations from triplicate cultures.
A differential dependence on Na for
putrescine and polyamine transport has led to postulate the existence
of multiple classes of polyamine carriers in some models (for review,
see Refs. 7, 58). As documented in the companion paper(37) ,
the physicochemical requirements for putrescine and spermidine uptake
are qualitatively very similar in ZR-75-1 cells, and both
processes are Na
-independent. On the other hand,
evidence for multiple putrescine and polyamine uptake systems has also
been based on the pattern of uptake inhibition by mutual
competitors(58, 59, 60) , whereas a single
class of carrier could account for di- and polyamine transport in other
models(12, 19, 61) . As shown by genetic
complementation(62) , although at least two different loci are
involved in putrescine and polyamine transport-deficient phenotypes, it
is not clear whether more than one structural transport gene is
involved.
On the basis of the kinetic parameters herein presented
for breast cancer cells, putrescine and spermidine separately behaved
as substrates for single, saturable transporters with a respective
affinity (putrescine < spermidine spermine) typical of most
mammalian cells(7) . The findings that (i) spermidine inhibited
putrescine uptake with a K
very close to its K
as a substrate, and that [ii] spermine
and APHHP, respectively, blocked putrescine and spermidine uptake with
closely similar K
values for both substrates, are
consistent with competition for a common carrier. On the other hand,
the K
of putrescine toward spermidine uptake was
much lower than expected from its own affinity as a substrate. Such a
behavior could reflect the existence of distinct high affinity
carriers, one being mainly responsible for polyamine transport, and the
second being shared by putrescine as well as polyamines, as proposed
for human endothelial cells(58) . However, this would predict a
biphasic inhibition of spermidine uptake by increasing concentrations
of putrescine, unlike the simple sigmoidal relationship observed (Fig. 2B). Moreover, Hill plots of spermidine uptake up
to 100 µM reproducibly yielded coefficients of unity (data
not shown), thus arguing against the existence of multiple classes of
spermidine transporters in ZR-75-1 cells. Therefore, if distinct
carriers exist for putrescine and polyamine transport, non-substrates
should inhibit the activity of a given transporter type through a
purely antagonistic interaction, e.g. polyamines should
inhibit putrescine binding to its specific carrier while not being
themselves substrates.
Previous studies have shown that aliphatic
compounds with two unhindered protonated amine centers separated by a
distance roughly corresponding to that found between the terminal amino
groups of spermidine is optimal for an efficient interaction with both
diamine and polyamine uptake systems in mammalian
cells(63, 64, 65) . This was confirmed
confirmed here by the relative ability of 1,6-diaminohexane,
1,7-diami-noheptane, and spermidine to inhibit putrescine uptake (Table 1). Moreover, the central imino group and one of the
terminal amino groups of spermidine and its homologs can be alkylated
without major loss of affinity for the transport system, as found with
APHHP (Table 1; (40) ). The inability of
3-cyclohexylpropylamine or 4-(3-aminopropyl)morpholine to compete for
spermidine uptake in ZR-75-1 cells further indicates that the
secondary amino group of APHHP is essential to preserve a high affinity
for the polyamine uptake system, while the sterically hindered tertiary
amino nitrogen is most likely dispensable in that respect. Indeed,
substantial structural modifications of the methylene backbone between
the two terminal amino groups of polyamines can be tolerated, as with
1,4-bis(3-aminopropyl)piperazine, but at the expense of substrate
affinity, as noted earlier for N-substituted
spermidine derivatives(66) . In addition to the key role of
properly distant and unhindered amine centers, specific hydrophobic
interactions must also largely contribute to the higher affinity of
spermidine for the polyamine transporter as compared to putrescine.
This is strongly suggested by the fact that N-alkylated
1,3-diaminopropane derivatives are much better competitors than
1,3-diaminopropane against putrescine uptake in ZR-75-1
cells(31) . Similar hydrophobic interactions likely explain the
increased ability of aliphatic amines to inhibit spermidine uptake with
a lengthening of the methylene carbon chain (Table 1; Refs. 40,
63).
A single putrescine/spermidine carrier model might reconcile
these observations if one postulates that spermidine, which differs
from putrescine by the addition of an aminopropyl group, binds to the
same recognition site as putrescine through hydrophobic as well as
electrostatic interactions unavailable for the diamine as a substrate.
Thus, a single putrescine/polyamine carrier could exhibit qualitative
differences in substrate binding characteristics toward the two classes
of substrates, which could account for difference between the K and K
of putrescine as a
substrate and a spermidine competitor, respectively. Interestingly,
cross-inhibition observed between putrescine and the herbicide paraquat
for their uptake in rat alveolar type II cells (67, 68) has been described as partial competitive
inhibition (43) resulting from a decrease in substrate affinity
caused by prior binding of the competitor to a distinct site on the
same carrier(69) .
Estrogens exert major mitogenic effects
in human breast cancer cells expressing functional cognate
receptors(70) . Several targets such as the progesterone
receptor, pS2, and procathepsin D genes have initially been identified
as being under specific estrogenic control(70) . More recent
evidence, however, indicates that polypeptide growth factors can induce
these same genes by activating common transcriptional elements involved
in the transduction pathway of estrogens(71, 72) . The
present data provide the first evidence that a membrane transport
system can also be under dual, synergistic regulation by estrogens and
a polypeptide growth factor. Moreover, the E- and
insulin-induced increase in polyamine uptake activity is an early event
in the cascade of events leading to exponential cell growth, which is
cooperatively stimulated by both hormones in ZR-75-1 as well as
other breast cancer cell lines(71) .
Although
putrescine and spermidine depletion blocks the mitogenic action of
insulin and E
, it does not interfere with the hormonal
stimulation of polyamine transport activity. The fact that the
hormone-dependent induction of the polyamine transport system was
independent of the cell growth status shows that it does not merely
result from the elevated ion fluxes accompanying mitogenic activation,
but may rather represent a novel, genuine target of estrogen and growth
factor action in mammary tumor cells. Whether the hormonal induction of
polyamine transport results from the increased activity of a constant
number of transporters or in the net addition of new carrier molecules
to the membrane is as yet undetermined, but insulin is known to control
the expression of the mammalian transporters GLUT-1 and GLUT-4 through
both mechanisms(73) .
Polyamine uptake in other tissue types
can be stimulated by peptide hormones, serum, and other mitogenic
stimuli(7) . However, with few exceptions(74) , most of
these effects were not clearly dissociated from the general trophic
effect of these agents. It is noteworthy that insulin and prolactin
induce polyamine uptake activity in the lactating mouse mammary
gland(21) , suggesting that the hormonal regulation of
polyamine transport may be a more general feature of mammary epithelial
cells. The dual and cooperative regulation of ornithine decarboxylase
expression and polyamine transport by estrogens and insulin
in human breast cancer cells may constitute a coordinate response to
increase the polyamine pool at early steps in the hormonal activation
of macromolecular synthesis. It should be noted that the rate of
polyamine biosynthesis is low, even under optimal growth conditions, in
most human breast cancer cell lines
examined(27, 31) .
Polyamine transport
in breast tumor cells may thus play a key role in polyamine homeostasis in vivo where amino acid precursors for endogenous polyamine
synthesis are likely more limiting than in the in vitro context. This idea is supported by the recent finding that
arginine limitation up-regulates polyamine transport in human
endothelial cells, an effect which is neither suppressed by ornithine
or putrescine(58) .
Interestingly, polyamine transport was
found to be under acute negative regulation by retinoic acid in
ZR-75-1 cells. Very few early biological responses to retinoids
have yet been reported in human breast cancer
cells(50, 51) . The rapid decay of spermidine uptake
activity induced by retinoic acid, which preceded any detectable effect
on ZR-75-1 cell growth, suggests that this membrane transport
system might be a proximal target of retinoid action in human breast
cancer cells. Since retinoic acid antagonizes growth factor action and
inhibits estrogen-dependent gene expression in human breast carcinoma
cells(50, 51) , down-regulation of polyamine uptake
might result from a similar effect on the expression of the
E- and insulin-regulated carrier. However, prior hormonal
induction of spermidine uptake activity markedly postponed any
detectable effect of retinoic acid on the latter parameter. Thus,
retinoic acid does not appear to interfere efficiently with the
transduction pathway synergistically activated by estrogens and
insulin, but might rather increase the turnover of the polyamine
carrier, which has a relatively long half-life(20) . Retinoic
acid has been reported to prevent the induction of putrescine transport
by blocking the G
/G
transition in initially
quiescent mouse hepatocytes(47) . It remains to be determined
whether retinoids may modulate polyamine transport by affecting cell
cycle distribution in breast tumor cells.
The present data clearly indicate that polyamine transport activity in human breast cancer cells is under stringent and rapid feedback inhibition by internalized polyamines, as in other mammalian cell types (17, 18, 19, 20, 21, 22) . The early feedback repression of polyamine transport in ZR-75-1 cells requires the de novo synthesis of a short-lived PTR protein, which represses carrier activity independently of its intrinsic transport capacity, which is metabolically stable (Fig. 7). Similar characteristics have been reported for the feedback inhibition of polyamine uptake in rat hepatoma cells and Chinese hamster ovary cells(22) . This PTR might be identical to the ornithine decarboxylase antizyme(75, 76) , as elegantly suggested by transfection experiments with expression vectors encoding a functional antizyme cDNA(24, 25) . Accordingly, antizyme, in addition to its role in directing the rate of ornithine decarboxylase degradation(76) , may function in the reversible down-regulation of polyamine carrier activity. In virtue of its short half-life and its rapid inducibility by polyamines, presumably at the translational level through a unique mode of ribosomal frameshifting(77) , antizyme would be especially well suited for the acute repression of polyamine transport.
Nevertheless, the feedback system regulating polyamine carrier activity in human breast cancer cells shows several novel regulatory features. First, polyamine carrier activity was not only repressed by the rapid induction of an unstable, antizyme-like PTR, but also by an as yet undescribed, more latent mechanism which does not require protein synthesis. Since preventing PTR synthesis deregulates polyamine accumulation, the down-regulation of polyamine carrier activity observed in CHX-treated cells might have resulted from a nonspecific, perhaps toxic effect of excessive polyamine levels. However, polyamine carrier activity was under feedback inhibition almost independently from de novo protein synthesis in DFMO-treated ZR-75-1 cells (Fig. 9) under conditions of controlled spermidine accumulation ( Table 3and Fig. 10). Thus, internalized polyamines can repress carrier activity through an additional mechanism which does not require de novo protein synthesis nor result nonspecifically from their potential cytotoxicity. Such a mechanism could be analogous to the trans-inhibition effect of internalized substrates on system A amino acid carrier activity, which is thought to result from the ``locking'' of the substrate-bound carrier complex in the cytoplasmic orientation(78) . Alternatively, internalized polyamines might activate a relatively stable modifier molecule such as casein kinase II, which is known to be strongly stimulated by polyamines(79) , and thus inhibit carrier activity through post-translational modifications.
Second, no evidence for an unstable PTR activity could be detected in ZR-75-1 human breast cancer cells without ongoing polyamine accumulation, unlike findings reported in rat and hamster cell lines (22) . Up-regulation of putrescine and spermidine uptake activity by CHX without prior incubation with exogenous substrate has also been reported in the fungus N. crassa(23) and in the lactating mouse mammary gland(21) , respectively. If intracellular polyamine levels can regulate PTR synthesis, the endogenous or free polyamine pool prevailing in ZR-75-1 cells would thus appear to be too low to sustain a basal repression of polyamine uptake activity. Therefore, the up-regulation of polyamine transport activity caused by DFMO in ZR-75-1 cells does not likely result from the suppression of a basal rate of PTR synthesis by polyamine depletion, but rather from an increase in the total polyamine transport capacity.
As a third, novel feature of the present model,
the inducibility of the putative PTR by internalized polyamines was
found to be markedly impaired by DFMO. A significant delay in PTR
induction due to the time needed to restore a normal, subthreshold
spermidine content in DFMO-treated cells is unlikely, since endogenous
spermidine level in control cells (1 nmol/mg DNA) represents only a
minor fraction of the amount accumulated within the first hour ( Fig. 8and Fig. 10). Since polyamines rapidly favor
antizyme translation(77) , sustained polyamine starvation could
decrease steady-state antizyme mRNA levels in ZR-75-1 cells,
assuming the identity of the PTR with antizyme. It should be noted that
antizyme mRNA expression is apparently constitutive in most vertebrate
tissues and does not respond to putrescine addition(75) ,
although its possible regulation by endogenous polyamines has not been
reported to our knowledge. Alternatively, the onset of PTR induction
could be delayed in DFMO-treated ZR-75-1 cells due to a
nonspecific effect of polyamine depletion on the overall rate of
protein synthesis (6) .
In addition to PTR induction and
trans-inhibition of carrier activity by internalized polyamines,
polyamine accumulation may also be regulated by other homeostatic
mechanisms beyond membrane carrier activity. This is suggested by the
fact that, although CHX did not markedly alter the kinetics of
down-regulation of spermidine carrier activity in DFMO-treated
ZR-75-1 cells, protein synthesis inhibition dramatically
increased spermidine, but not putrescine accumulation. Thus, although
DFMO impairs PTR induction and up-regulates polyamine transport
activity, spermidine overaccumulation in DFMO-treated cells is still
largely prevented by at least one additional mechanism requiring
protein synthesis. For instance, the synthesis of a protein regulating
polyamine efflux might be induced when intracellular polyamine levels
exceed a given threshold, which could account for a decrease in net
substrate accumulation despite ongoing uptake. However, our preliminary
results strongly indicate that the rate of polyamine efflux is not
affected by the extent of substrate accumulation (data not shown). An
alternative candidate might be spermidine/spermine N-acetyltransferase, which is rapidly induced by
polyamines and analogs, but much less so by putrescine, and is likely
involved in the homeostasis of polyamine pools(80) , as
suggested by the low amounts of N
-acetylspermidine
formed in DFMO-treated cells incubated with spermidine. Whether
spermidine/spermine N
-acetyltransferase induction
can solely account for the post-carrier regulation of polyamine
accumulation remains to be determined by comparison with
acetylation-resistant polyamine analogs.
Upon inhibition of de
novo protein synthesis, ZR-75-1 cells accumulate
3-6-fold higher levels of various substrates of the polyamine
transport system, as in other eukaryotic cells(22) . There is
now substantial evidence that excessive intracellular polyamine levels
are highly toxic to eukaryotic
cells(54, 55, 56, 57) . In fact,
polyamine overaccumulation leads to rapid cell death by apoptosis in
ornithine decarboxylase-overexpressing L1210 cells, without
prior oxidation of the accumulated substrate(57) . A similar
defect in the feedback regulation of spermidine uptake leading to
lethal polyamine accumulation has been reported in ornithine
decarboxylase-overproducing HTC cell variants(55) . If antizyme
is indeed the polyamine-induced PTR, the intriguing connection between
high ornithine decarboxylase expression and an altered feedback
regulation of polyamine transport might find an explanation in an
abnormally high mobilization of antizyme molecules by the large pool of
ornithine decarboxylase, thus decreasing their availability for
feedback transport inhibition. Thus, the present data support the
notion that multiple mechanisms of feedback inhibition might be needed
to prevent apoptosis or other forms of cytotoxicity (57) caused
by polyamine overaccumulation. Quite notably, putrescine induced PTR
protein activity only after a long lag period and caused virtually no
down-regulation of spermidine uptake in the presence of CHX. Thus, the
diamine is much less active than spermidine or spermine both as an
inducer of PTR and as a trans-inhibitor of polyamine uptake, and the
present data suggest that its limited repressing effect might in fact
be due to its conversion to spermidine. It is noteworthy that
putrescine overaccumulation caused by inhibition of S-adenosylmethionine decarboxylase(53) , ornithine
decarboxylase overproduction(52) , or the relief of feedback
inhibition of putrescine uptake (55) is not overtly toxic,
perhaps with some exceptions (56) or may even be beneficial in
certain physiological situations(52) . The comparatively low
cytotoxicity of putrescine could therefore account for its much weaker
activity in feedback transport inhibition.
The present finding that
DFMO-induced polyamine depletion strongly interferes with the induction
of an unstable PTR by internalized polyamines, but not with the overall
regulation of net polyamine accumulation, raises new questions as to
the exact role of this putative protein in the regulation of polyamine
accumulation. PTR induction and, at later stages, trans-inhibition by
internalized substrates might mostly govern the initial velocity of
polyamine transport, with post-carrier mechanisms dependent on de
novo protein synthesis such as spermidine/spermine N-acetyltransferase induction for the final
adjustment of the size of the polyamine pool.