Kinetics of cyclocreatine and
Na+ cotransport in human
breast cancer cells: mechanism of activity
Nimrod
Maril1,
Hadassa
Degani1,
Edna
Rushkin1,
A. Dean
Sherry2, and
Mildred
Cohn3
1 Department of Biological
Regulation, Weizmann Institute of Science, Rehovot 76100, Israel;
2 Department of Chemistry,
University of Texas at Dallas, Richardson, Texas 75083-0688; and
3 Department of Biochemistry and Biophysics,
University of Pennsylvania, Philadelphia, Pennsylvania 19104-6089
 |
ABSTRACT |
The growth-inhibitory effect of cyclocreatine
(CCr) and the kinetics of CCr and
Na+ cotransport were investigated
in MCF7 human breast cancer cells and its adriamycin-resistant subline
with use of 31P- and
23Na-NMR spectroscopy. The
growth-inhibitory effect in the resistant line occurred at a lower CCr
concentration and was more pronounced than in the wild-type line. This
correlated with an ~10-fold higher affinity of CCr to the transporter
in the resistant line. The passive diffusion coefficient of CCr was
also higher in the resistant line by three- to fourfold. The transport
of CCr was accompanied by a rapid increase in intracellular
Na+. This increase was found to
depend on the rate of CCr transport and varied differently with CCr
concentration in the two cell lines. It is proposed that the
cotransport of CCr and Na+
followed by increased Na+
concentration, together with the accumulation of the highly charged phosphocyclocreatine, are responsible for cell swelling and death.
phosphorus-31 nuclear magnetic resonance; sodium-23 nuclear
magnetic resonance; cell perfusion
 |
INTRODUCTION |
CYCLOCREATINE
[1-carboxymethyl-2-iminoimidazolidine (CCr)], a creatine
(Cr) analog, has been shown to act as an anticancer agent in a large
range of cell culture systems and in tumors implanted subcutaneously in
rats and mice (25, 31, 34, 41, 47). The mechanism of cell death by CCr
in C6 glioma cells appeared to be
related to the accumulation of phosphocyclocreatine (PCCr) and cell
swelling (41, 42). However, the detailed mechanism by which CCr
enhances cell death is unknown.
CCr and Cr are transported into the cells and are rapidly
phosphorylated by the creatine kinase (CK) reaction to form PCCr and
phosphocreatine (PCr), respectively, according to the following scheme
|
(1)
|
where CCri is intracellular
CCr, CCre is CCr in the perfusion
medium, kCCr is
the apparent first-order rate constant of CCr transport, and
k1 and
k
1 are the
forward and backward rate constants of the CK reaction, respectively
(33). The CK reaction is a dead-end reaction and, in general, serves to
regulate energy in the cell (for review see Ref. 52). Among all Cr
analogs, CCr is the best substrate of the CK reaction by the
Vmax/Km
criterion (where
Vmax is maximal
reaction velocity and
Km is
Michaelis-Menten constant) (3, 29, 33, 50, 54). The rate of ATP
generation from PCCr is 160-fold slower than the rate of ATP generation
from PCr, and the reverse reaction is 5-fold slower (3). This resulted in an ~30-fold higher equilibrium constant of the CK reaction when
CCr served as a substrate of CK (scheme
1). Thus, when rats are fed CCr in the diet, at
equilibrium 98% of the total CCr in various tissues is present as PCCr
(50). In the body, Cr is predominantly synthesized in the pancreas,
liver, and kidney and is then exported to the circulating blood and
transported into the cells (48, 49). The transport has been well
studied, and recently the human Cr transporter has been cloned (36) and
characterized after expression in
Xenopus oocytes (12).
Although there is no universal detailed mechanism proposed by the
various investigators, all have revealed an
Na+-dependent component (12, 13,
27, 28, 35). In cell culture studies, two saturable processes or a
combination of a saturable transport component and a diffusion
component were identified (13, 27, 28, 35). In
31P-NMR studies of Cr uptake in
liver expressing CK, a pH-dependent saturable Michaelis-Menten type of
kinetics was described (32). By monitoring PCCr with use of
31P-NMR, active (Michaelis-Menten)
and diffusion mechanisms were found to contribute to the transport of
CCr in ex vivo studies of uterine tissue (45). In
31P-NMR studies of
C6 glioma cells, the active
component of CCr transport was characterized and its
Na+ dependence was demonstrated
(41).
31P-NMR detects not only the PCr,
but also other phosphorus-containing metabolites. Of special
significance is the level of nucleoside triphosphates (NTPs) and
internal Pi, which reflect the
energy state of the cells (15).
23Na-NMR of perfused cells detects
the overlapping signals of the intra- and extracellular
Na+. However, the use of an
anionic relaxation or frequency shift reagent results in the removal of
this degeneracy (14, 19, 40). The anionic chelate complexes of
paramagnetic lanthanides, particularly
Dy3+ and
Tm3+, on binding to the
Na+, cause a resonance hyperfine
shift of the
23Na+
signal. Because the anionic paramagnetic shift reagents do not penetrate the cell membrane within the time scale of NMR measurements (1-2 days), only the extracellular
Na+ ions experience the resonance
shift. Thulium
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate)
(TmDOTP5
) has been shown
to be stable in blood and was successfully used as a shift reagent to
detect the intracellular Na+ in
animal experiments (4, 5, 30, 43). In early in vivo studies with this
shift reagent (1), it was toxic at high levels; in later studies (4, 5,
43), with proper anesthesia, it was well tolerated by rats. At the
concentration used here (10 mM) for cell culture studies,
TmDOTP5
did not affect cell
energetics (i.e., NTP concentration) and appeared to be nontoxic.
The goal of the present studies was to elucidate the mode of action of
CCr as an anticancer agent. We investigated and compared the effect of
CCr on the growth rate and the morphology of MCF7 wild-type (WT) and
the adriamycin-resistant clone MCF7
AdrR human breast cancer cell
lines (16). 31P- and
23Na-NMR were applied to determine
noninvasively the kinetics of CCr and
Na+ cotransport, respectively, in
these cells. The higher sensitivity of the MCF7
AdrR clone to CCr than that of the
WT clone was correlated with the kinetic parameters of the cotransport
of CCr and Na+ in the two cell lines.
 |
MATERIALS AND METHODS |
Cell culture.
MCF7 and MCF7 AdrR human breast
cancer cells were kindly supplied by Dr. Lippman (National Institutes
of Health, 1987 and 1995, respectively). MCF7 cells were routinely
cultured in DMEM supplemented with 6% FCS (Biological Industries,
Beit-Haemek, Israel) and 0.1% combined antibiotics (Bio-Lab,
Jerusalem, Israel) containing penicillin (200,000 U/ml), streptomycin
(200,000 µg/ml), and neomycin (10,000 µg/ml). MCF7
AdrR cells were cultured routinely
with a 1:1 mixture of minimum essential medium and F-12 medium
supplemented with 10% FCS, 0.1% combined antibiotics, and additional
glucose (1 g/l), sodium bicarbonate (1 g/l), choline chloride (50 mg/l), and inositol (30 mg/l). The final growth medium contained a
negligible amount (~22 µM) of Cr, which was present only in the
serum (2).
In cell experiments with Cr (Sigma Chemical) or CCr [synthesized
by the method of Wang (53)], stock solutions of medium containing
each metabolite at 40 mM were used to reach a final concentration of
0.3-19 mM.
Growth studies.
Cells were plated in 24-well dishes (Nunc) at a density of ~1.5 × 104 cells/well in 1 ml of
culture medium and placed in a 37°C, 5% CO2 humidified incubator. After 48 h the medium was replaced by fresh medium containing various
concentrations of CCr or Cr. Growth of cells was followed with time by
cell counting with an inverted light microscope by use of a
hemocytometer. Inhibition of cell growth was calculated from the number
of cells in treated (Nt) and control (Nc) samples in the same time
period, yielding percent reduction of cell number from control of (Nc
Nt)/Nc × 100.
CK assays.
Assays were performed on the supernatant of sonicated cells. CK
activity was measured spectroscopically at 340 nm with use of a coupled
assay, as previously described (44). Protein was determined by the
Bradford method (7). CK activity is given as the mean ± SE of three
independent experiments, in units of micromoles per minute per
milligram protein.
Morphological assessment of cell cultures.
MCF7 and MCF7 AdrR cells were
cultured in petri dishes as described above. Two days after seeding,
the standard growth medium was replaced every 48 h by fresh medium for
the controls and, in addition, 20 mM CCr for the treated cells. Both
cell cultures were examined at 24-h intervals with an inverted light
microscope to monitor changes in the diameter of the cells and to
calculate the mean area (n = 20) with
the assumption of a circular shape. The change in area of the cells
during treatment with CCr is reported as the ratio of the mean area
before to that after treatment plus or minus the uncertainty.
Cell cultures during NMR experiments.
For NMR studies, cells (MCF7 and MCF7
AdrR) were cultured on
agarose-polyacreloin beads (250-500 µm) or on Biosilon
polystyrene beads (160-300 µm, Nunc) with the growth medium
described above by following procedures described previously (37). The
cells grown on beads (2-2.5 ml) were placed in a 10-mm sterile NMR
tube and perfused with oxygenated medium at 36 ± 1°C, as
described previously (15). In the
31P studies the cells were
perfused with DMEM containing 10% FCS and combined antibiotics. In the
23Na studies, cells were perfused
with Na+-free growth medium as
described above containing the shift reagent Na3H2TmDOTP · 3NaCl
at 10 mM (8) and additional NaCl to reach a final concentration of 155 mM Na+.
NMR experimental parameters.
NMR spectra were recorded with a Bruker AM-500 spectrometer with use of
a multinuclei broad-band probe.
31P-NMR spectra were recorded at
202.5 MHz by acquiring 900 transients with 45° pulses, 59 µs
between the pulse and the acquisition (DE), 2-s repetition time, and
0.38-s acquisition time, with a continuous composite pulse proton
decoupling of ~1 W. The chemical shift of
31P signals was referenced to
-NTP at
10.03 ppm. Concentrations were calculated from the
integrated areas of the corresponding signals, with the integrated area
of medium Pi (1 mM) used as reference and correction for saturation effects. Analysis of the 31P spectra was performed with a
standard Bruker software package, XWIN-NMR, with application of
integration and line shape-fitting modes.
23Na-NMR spectra were recorded at
132 MHz by acquiring 2,000 transients with 90° pulses, DE of 86 µs, 0.47-s repetition time, and 0.27-s acquisition time.
Concentration of the intracellular Na+ was calculated from the
integrated area of its signal referenced to the integrated area of the
medium Na+ signal (155 mM, 100%
NMR visibility) with the assumption of 40% NMR visibility of the
intracellular Na+. In most cells
the intracellular Na+ signal is
only 40% visible by NMR compared with the extracellular Na+ signal, which is 100% visible
(6). When DE is very short, a full visibility of the intracellular
Na+ might be regained. However,
for the DE value applied here, the assumption of 40% visibility is
justified. The energy state of the cells and the extent of PCCr
accumulation in the 23Na
experiments were characterized by recording
31P spectra at the beginning and
end of the experiments with the cells perfused with the standard growth
medium in the absence of the shift reagent. The integrated areas of the
23Na signals were obtained with a
standard Bruker software package, XWIN-NMR.
 |
RESULTS |
Cell growth and CK activity.
Studies of the growth of MCF7 and MCF7
AdrR cells in the presence of
various concentrations of CCr indicated a dose-dependent growth
inhibition by this drug (Fig. 1,
A and
B). The sensitivity to
the drug was different in each cell line. In the MCF7 line, cells
continued to grow in the presence of CCr, but at a slower rate than
untreated cells (Fig. 1A). For
example, after 5 days in the presence of 20 mM CCr, the number of cells
relative to day 0 increased by 28%,
but the percent reduction of cell number from control was 61%. On the
other hand, in MCF7 AdrR cells,
CCr markedly inhibited growth (Fig.
1B); after 5 days in the presence of
20 mM CCr, the number of cells relative to day
0 actually decreased by 23% and the percent reduction
from control was 93%. In a similar study with Cr in MCF7
AdrR cells, contrary to the
behavior of CCr, only a marginal inhibition of growth was observed
(Fig. 1C). At a very high
concentration of Cr (40 mM) and after 7 days of treatment, reduction of
MCF7 AdrR cell number relative to
control was 52%.

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Fig. 1.
Growth curves of MCF7 and MCF7
AdrR cells in presence of
cyclocreatine (CCr) and creatine (Cr).
A: MCF7 cells with CCr;
B: MCF7
AdrR cells with CCr;
C: MCF7
AdrR cells with Cr. Cells were
cultured under standard conditions ( ) or treated with 5 ( ), 10 ( ), 20 ( ), and 40 ( ) mM CCr or Cr. Each point represents mean;
error bars, SE (n = 4-6).
|
|
The activity of CK was 0.026 ± 0.009 and 0.069 ± 0.032 µmol · min
1 · mg
protein
1 in MCF7 cells
(n = 3) and in the resistant cell line
(n = 3), respectively. Thus the mean
CK activity was almost threefold higher in the resistant than in the WT cells.
Cell swelling.
MCF7 AdrR cells grown as a
monolayer in the presence of 20 mM CCr exhibited distinct morphological
changes and marked cell swelling (Fig. 2).
After 6 days of treatment the mean cell area increased by 3.0 ± 0.2-fold (ratio of means ± uncertainty) compared with control
cells. The WT cells in the presence of the same concentration of CCr
(20 mM) exhibited less swelling, and after 6 days of treatment their
mean area increased by 1.5 ± 0.5-fold (ratio of means ± uncertainty). At this concentration the transport of CCr is dominated by a diffusion mechanism, for which the rate is about fourfold higher
in the resistant line than in the WT (see below).

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Fig. 2.
Light-microscopic views of MCF7
AdrR cells grown for 6 days under
standard conditions (A) and in
presence of 20 mM CCr (B). Cellular
swelling is evident in B. Arrows in
A and B show typical cells.
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CCr transport measured by 31P-NMR.
31P spectra of both cell lines
exhibited signals of phosphorus in soluble metabolites that are present
at millimolar concentrations (Figs.
3B and
4B),
including NTPs, PCr, and Pi
(intra- and extracellular), all associated with cell energetics (11).
The PCr signal was more intense in MCF7
AdrR than in WT cells (Figs.
3B and
4B). In addition, signals due to
phosphocholine, phosphoethanolamine, and uridine diphosphosugars were
detected. Glycerophosphoethanolamine and glycerophosphocholine were
present at low concentrations in WT cells (Fig. 3) and were below
detection in the resistant clone (Fig. 4).

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Fig. 3.
31P-NMR spectra of MCF7 cells
perfused at 37°C under standard conditions without
(B) and with 3.6 mM CCr for 5 h
followed by 9.4 mM CCr for 3 h (A).
Cells were cultured on microcarrier beads, and spectra were acquired
for 30 min. PE, phosphoethanolamine; PC, phosphocholine; int,
intracellular; ext, extracellular; GPE, glycerolphosphoethanolamine;
GPC, glycerolphosphocholine; PCr, phosphocreatine; PCCr,
phosphocyclocreatine; NTP, nucleoside triphosphate; UDPS, uridine
diphosphosugars. Spectra were processed with a line broadening of 20 Hz.
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Fig. 4.
31P-NMR spectra of MCF7
AdrR cells perfused at 37°C
under standard conditions without
(B) and with 0.33 mM CCr for 12.5 h
(A). Cells were cultured on
microcarrier beads. See Fig. 3 legend for recording
parameters and definition of abbreviations.
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After addition of CCr to the perfusion medium, a distinct signal from
PCCr (
2.34 ppm) close to that of PCr appeared in the 31P spectrum (Figs.
3A and
4A). Initially, it was possible to
resolve PCr and PCCr signals. During this period the PCr signal
intensity did not change. After several hours of perfusion with CCr, as PCCr became predominant, the two signals could not be resolved. However, with the assumption that PCr is constant, the increase of the
combined signal of PCr and PCCr reflected PCCr accumulation.
The mechanism and kinetic parameters of CCr transport into MCF7 and
MCF7 AdrR cells were determined by
sequentially increasing CCr concentration in the perfusion medium and
monitoring PCCr with 31P-NMR. The
rate of increase in the PCCr signal is limited by the rate of transport
of CCr or by the rate of the phosphorylation through the CK reaction.
The following analysis of previous kinetic data supports the assumption
that transport of CCr is the rate-limiting step. The CK-catalyzed rate
of PCr synthesis from Cr in T47D human breast cancer cells, which
demonstrate a CK activity and ATP-to-PCr ratio similar to MCF7
AdrR cells, was 0.2 mM/s (38).
Inasmuch as the rate of PCCr synthesis is fivefold slower than the rate
of PCr synthesis (3), the estimated CK flux to form PCCr in MCF7
AdrR cells is ~0.04 mM/s.
Similarly, a threefold lower flux is estimated for the CK reaction in
the WT cells on the basis of their lower CK activity (see above). The
Michaelis-Menten
Vmax derived from the rates of PCCr increase (Eq. 2)
in both cell lines was on the order of 3 fmol · cell
1 · h
1
(Table 1), which is equivalent to ~0.0005
mM/s (average cell volume of 1,500 µm3). This rate is more than
one order of magnitude slower than the above-estimated rates of PCCr
synthesis by CK and can be attributed only to the transport. Therefore,
the rate of PCCr increase served to measure CCr transport by applying
the zero trans method (46).
The initial change with time in the intensity of the PCCr signal was
linear. The apparent initial rate
(Vappinit) as a function of
CCr concentration was best fitted to Eq. 2 with two mechanisms of transport:
1) saturable, Michaelis-Menten-like active transport and 2) passive
diffusion (Fig. 5)
|
(2)
|
where
[PCCr] is PCCr concentration, S is CCr
concentration in the medium,
Vmax is the
maximum velocity,
Km is the
Michaelis Menten constant, and
Kd is the passive
diffusion constant. The resultant kinetic parameters are
summarized in Table 1. The results clearly demonstrated that the
Km of active
transport in the resistant line was an order of magnitude lower than
that in the WT cells, whereas
Vmax was similar.
In addition, Kd
was fourfold higher in the resistant line.

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Fig. 5.
Kinetic parameters of CCr transport into MCF7 cells.
V, apparent initial rate; S,
CCr concentration; K, apparent initial
rate/CCr concentration (K = V/S). Results were
obtained from 31P-NMR spectra that
monitored time course of signal intensity of PCCr signal with increased
CCr in medium. Curves were obtained by fitting data to
Eq. 2, consisting of an active
transport component and a passive diffusion component.
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CCr and Cr effect on intracellular
Na+ content.
The transport of Na+ concomitant
with the transport of CCr was monitored by changes in the intracellular
Na+ concentration with
23Na-NMR.
23Na spectra of WT and resistant
cells perfused with medium containing 10 mM
TmDOTP5
as a shift reagent
revealed two separate signals (Fig. 6). The extracellular Na+ signal was
shifted downfield by ~13 ppm from the intracellular Na+ signal, arbitrarily assigned
to 0 ppm, making it possible to detect and quantitate the signal of
each Na+ compartment separately.
The shift of the extracellular Na+
signal decreased with time in parallel to the decrease in the medium pH
(Fig. 6), as previously reported (23). After 15 h the shift of the
extracellular Na+ was ~10 ppm
(Fig. 6A); however, this shift was
still large enough for complete resolution of the two signals. To
examine the stability of the steady-state
Na+ level, cells were initially
perfused for 9 h in the presence of 10 mM
TmDOTP5
. During this period
there was no change in the Na+
content, confirming that neither the NMR conditions nor the presence of
10 mM TmDOTP5
in the
perfusion medium affected the intracellular
Na+ content (Fig.
7A).

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Fig. 6.
23Na-NMR spectra of MCF7
AdrR cells perfused at 37°C
with medium containing 10 mM
TmDOTP5 .
B: before exposure to CCr.
A: after 15 h of perfusion with
medium containing 10 mM CCr. Cells were cultured on microsphere beads.
Spectra were recorded by accumulating 2,000 transients (17 min), with
90° pulses and 0.2-s repetition time, and processed with a line
broadening of 3 Hz. ×16, 16-Fold magnification.
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Fig. 7.
Intracellular Na+ content of MCF7
AdrR in presence of 10 mM Cr
(A) or 10 mM CCr
(B). Cells were cultured and
perfused with medium containing 10 mM
TmDOTP5 .
Na+ content was determined from
integrated area of intracellular
23Na-NMR signal.
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|
Addition of CCr to the perfusion medium induced an increase in the
intracellular Na+ signal (Fig. 6).
The time course of the rapid increase of the intracellular
Na+ content in MCF7
AdrR cells perfused with 10 mM CCr
is shown in Fig. 7B with a rate of 3.7 ± 0.7 fmol · cell
1 · h
1.
At the beginning of the experiment the intracellular
Na+ content was 77 fmol/cell.
Seventeen hours after CCr addition, the intracellular
Na+ content was 155 fmol/cell.
Then the level of the intracellular signal started to decrease (Fig.
7), paralleling cell death, as confirmed by
31P spectra obtained at the end of experiments.
When Cr was added to the perfusion medium of MCF7
AdrR cells, a very slow increase
in the intracellular Na+ (relative
to the increase in the presence of CCr) of 1.5 ± 0.3 fmol · cell
1 · h
1
occurred. After 7 h the initial
Na+ level (78 fmol/cell) reached a
steady state of 90 fmol/cell, which persisted until the end of the
experiment (Fig. 7A).
The rates of intracellular Na+
accumulation and of CCr transport have been compared to determine
whether a correlation exists between them. The rates of CCr transport
via the active transport mechanism in the two cell lines were
calculated on the basis of the
Vmax and
Km values found
in 31P-NMR transport experiments
(Table 2). In the resistant line at 1 and
10 mM CCr, the transport rates are close to the saturation rate.
However, in the WT line, the rate at 10 mM CCr is close to saturation,
whereas at 1 mM the transport rate is ~3.5-fold slower. Therefore, we
have chosen to measure the intracellular Na+ accumulation rates in both
cell lines at 1 and 10 mM CCr (Fig. 8) and
determined the ratio of these rates for both cell lines (Table
3). This ratio is independent
of the assumption of 40% Na+
visibility in the intracellular compartment and of the experimental variability in the initial Na+
concentration (65-100 fmol/cell). The ratios of intracellular Na+ accumulation rates in the
presence of 10 mM to those in the presence of 1 mM CCr in the perfusion
medium were similar to the ratios of the transport rates of CCr through
the active transport component at 10 mM to those at 1 mM CCr (Tables 2
and 3).

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Fig. 8.
Na+ accumulation in MCF7
(A and
C) and MCF7
AdrR cells
(B and
D) in presence of 10 mM CCr
(A and
B) or 1 mM CCr
(C and
D). Cells were cultured and perfused
in NMR spectrometer. Na+ content
was determined from integrated area of intracellular
23Na-NMR signal.
Nain, intracellular
Na+.
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 |
DISCUSSION |
The mechanism by which CCr could exert its anticancer activity was
investigated in two lines of MCF7 human breast cancer cells: the WT
cell and its adriamycin-resistant subline. CCr induced a time- and
dose-dependent inhibition of growth in the two cell lines. The
resistant cells exhibited a much higher sensitivity to CCr with a
distinct cytotoxic effect.
In general, cancer cells with resistance induced by one therapeutic
agent become resistant to other drugs or are unaffected by them; rarely
do they become more sensitive to structurally unrelated agents, as in
the case of CCr with MCF7 AdrR
cells presented in this report. A similar phenomenon of increased toxicity in MCF7 AdrR cells has
been observed with 2-deoxyglucose (2-DG) (20, 21). In that study the
greater toxicity in the resistant cells was ascribed to many factors,
especially the faster depletion of ATP to form phospho-2-deoxyglucose
(P-DG). The increased rate of accumulation of P-DG in the
resistant cells relative to WT cells as monitored by
31P-NMR was ascribed to faster
phosphorylation rather than faster transport into the cell. Another
example of an anticancer agent to which resistant cells are more
sensitive than the parental line is ardeemins; no mechanism was
suggested (9). In the present study we attempted to elucidate the cause
of the toxic activity of CCr by pinpointing the source of the
quantitative differences between the WT and the resistant cells.
The accumulation of PCCr in the cell is governed by two processes:
transport of CCr into the cell and its reversible phosphorylation by
ATP catalyzed by CK. The essential presence of CK for the cytotoxic activity of CCr was demonstrated previously in a study of a large number of cancer cell lines (25). However, when CK reaches high enough
activity, the phosphorylation is no longer rate limiting, and the
transport capacity may become dominant in determining CCr cytotoxicity.
The CK level in both cell lines, as noted in RESULTS, is sufficiently high not to
be rate limiting. Furthermore, the two- to threefold higher CK activity
in the resistant line cannot explain the markedly higher sensitivity to
CCr of the resistant cells. This was further supported by measurements
of CK activity and cell growth inhibition by CCr in T47D cells (not
shown). Although in these cells the CK activity (0.058 ± 0.031 µmol · min
1 · mg protein
1) was close to
that of the MCF7 AdrR clone, their
sensitivity to CCr was very low, even less than that of MCF7 WT cells.
In contrast to the ~2.5-fold difference in CK activity, we have found
that the Km of
active transport of CCr in the resistant clone is 10-fold lower than
that in the WT cells. With a 10-fold higher affinity of the transporter
for its substrate (CCr) in the resistant clone, a higher sensitivity of
these cells is obtained at concentrations of CCr below the
Km of active
transport. The kinetic studies carried out with
31P-NMR of perfused cells also
yielded the Vmax
of CCr transport and the coefficient of the diffusion component of the
transport (Table 1). At concentrations of CCr high above the
Km (>10 mM), the active transport proceeds at a
Vmax that is
similar for both cell lines. However, at these concentrations the
fourfold higher diffusion coefficient in the resistant clone may also
contribute to the difference in the sensitivity of the cells to CCr.
The higher diffusion rate leads to a more rapid increase in
intracellular CCr, which is rapidly converted to PCCr. Because the
intracellular equilibrium concentration ratio of PCCr to CCr is high
(3, 29, 54), CCr continues to diffuse into the cell, causing an accumulation of PCCr and an osmotic imbalance, which together with
Na+ accumulation by the active
transport component leads to cell swelling and death.
The active transport, for which
Km values were
found to differ markedly between the two cell clones, has been
previously shown to be Na+
dependent (13, 18, 27, 28, 35, 41, 45). Therefore, we have considered
the role Na+ may play in the
anticancer activity of CCr. In the initial studies the role of
Na+ was indirectly characterized
by measuring changes in the rate of Cr or CCr transport at various
Na+ concentrations. The most
direct method of evaluating the role of
Na+ is to follow the intra- and
extracellular Na+ by
23Na-NMR simultaneously with PCCr
by 31P-NMR during the CCr
transport process into MCF7 and MCF7
AdrR cells.
In the presence of CCr in the perfusing medium, a linear intracellular
accumulation of Na+ with different
rates was observed in both cell lines. The agreement between the rates
of CCr-induced intracellular Na+
accumulation, observed and anticipated from the respective kinetic parameters of CCr transport in the two cell lines, contributes strong
additional evidence for a cotransport mechanism. A similar cotransport
process was described for glucose (39) and for amino acid transport
(10, 22).
In the resistant clone, because of the low
Km of CCr
transport (0.4 mM) the rates of
Na+ accumulation at 1 and 10 mM
CCr were close. In the WT cells, where
Km was higher (4 mM), a marked difference in the rates of Na+ accumulation, at the two CCr
concentrations, was measured. The similarity of the rates of
Na+ accumulation at 10 mM in the
two cell lines was consistent with the similarity of the
Vmax values. In
the presence of Cr, a slow increase in the
Na+ concentration to a
steady-state level (90 fmol/cell) was observed, unlike the rapid
intracellular accumulation of Na+
to much higher concentrations (160 fmol/cell) in the presence of CCr.
This correlates with the sensitivity of the cells to CCr, and not to
Cr, the natural substrate.
The Na+ accumulation could occur
as a result of inhibition by a direct binding of CCr and/or PCCr to the
Na+-K+-ATPase,
as in the case of ouabain (26). Alternatively, the higher
Na+ concentration, which
accompanies CCr transport, due to the more favorable CK equilibrium
constant, may saturate the
Na+-K+-ATPase
pumps so that the efflux becomes slower than the influx. Undoubtedly, a
contributory mechanism is an indirect inhibition of
Na+ efflux caused by the inability
of the cells to pump out the Na+
for energetic reasons. ATP needed to operate the
Na+ pump can be regenerated by the
CK reaction from PCr by a direct functional coupling between the CK
reaction and the Na+ pump (17, 24,
51). However, in the presence of PCCr, the regeneration of ATP is two
orders of magnitude slower, and the energy supply may not be sufficient
to fully activate the pump.
In summary, it is proposed that the impairment of cells in maintaining
Na+ homeostasis in the presence of
CCr (unlike Cr), leading to a net accumulation of
Na+ together with the accumulation
of the highly charged PCCr, is responsible for cell swelling and death.
The difference in sensitivity of various cell lines to CCr may be
ascribed to differences in their kinetic parameters of active
cotransport of CCr and Na+.
 |
ACKNOWLEDGEMENTS |
We thank Prof. A. M. Kaye (Weizmann Institute of Science) for
assistance with the CK studies and helpful discussions.
 |
FOOTNOTES |
This study was supported by the German-Israel Foundation for Scientific
Research and Development.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Degani, Dept.
of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel (E-mail: cidegani{at}weizmann.weizmann.ac.il).
Received 3 March 1999; accepted in final form 21 June 1999.
 |
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