Download Mechanism of Cyclophosphamide Transport by

[CANCER RESEARCH 34,3274—3282,
December1974]
Mechanism of Cyclophosphamide Transport by
L5178Y Lymphoblasts in Vitro'
GeraldJ. Goldenberg,2H. BernardLand,and DouglasV. Cormack
Department of Medicine, University ofManitoba, and The Manitoba Institute ofCeIl Biology fG. J. G., H. B. L.J and Department ofPhysics, The
Manitoba Cancer Treatment and Research Foundation and the Department ofMedical Microbiology, University ofManitoba [D. V. C.J , Winnipeg,
Manitoba, R3E 0V9, Manitoba, Canada
INTRODUCTION
SUMMARY
Mechanism of transport
of the alkylating agent cyclophos
phamide-' 4C was investigated
in L5 178Y lymphoblasts
in
vitro. A time course of cyclophosphamide
uptake showed a
rapid, initial phase, probably due to binding of drug to the cell
surface. Subsequent uptake into the cells was carrier mediated
and consisted of two components. Analysis of cyclophos
phamide uptake over a 40-fold range of drug concentration
showed biphasic kinetics with evidence of saturation only at
low drug concentrations whereas, at high drug levels, uptake
occurred by a second transport system that was technically
nonsaturable.
@
@
After correction
for binding
and the interaction
of two-component transport, kinetic parameters for low-dose
transport consisted of a Michaelis constant Km (mean ±S.E.)
of 0.39 ±0.03 mM and a transport capacity Vmax of 0.49 ±
0.07 X l0'
moles/mm/cell. At high-dose cyclophosphamide
transport, the apparent K@ was 75 ±29 mM , and the Vmax
was 49 ±14 X l0'
moles/mm/cell. Both low- and high-dose
cyclophosphamide transport were temperature sensitive and
partially dependent on sodium. In addition, low-dose transport
was inhibited by oligomycin and cyanide. Other alkylating
agents and several naturally occurring substrates did not
inhibit
cyclophosphamide
transport
; thus, a native
agents.
Evidence that low-dose cyclophosphamide transport was
mediated by a facilitated diffusion process was that uptake
obeyed saturation kinetics, was temperature and sodium
dependent,
was partially
dependent
on metabolic
energy,
and
cell/medium concentration gradients did not exceed unity.
Although high-dose drug uptake failed to show saturation
kinetics, the demonstration
of temperature
and sodium
dependence also suggested that high-dose uptake may be
carrier mediated.
Cyclophosphamide uptake by chick embryo liver cells was
examined also ; uptake was temperature sensitive and exhibited
biphasic kinetics similar to that observed in L5 178Y cells,
suggesting a similar mechanism of drug transport in normal
liver and leukemic cells.
I This
work
was
supported
by
a
grant
from
the
National
Cancer
Institute of Canada.
2 Clinical
Research
Associate
of
the
National
Cancer
Canada.
Received June 3, 1974; accepted August 27, 1974.
3274
coma cells in vitro (19). Choline,
a close structural
analog of
HN2, has been identified as the native substrate for the HN2
transport system (20). It was also shown that other alkylating
agents, including intact and enzyme-activated cyclophospha
mide, did not inhibit HN2 transport, suggesting independent
transport mechanisms for these agents (21).
Accordingly, an investigation was undertaken of the
mechanism
of transport
of cyclophosphamide
by L5 178Y
lymphoblasts in vitro. Transport studies of alkylating agents
may be obscured by binding of drug through alkylation
reactions. This problem has been circumvented by the use of
hydrolyzed derivatives, which are inactive as alkylating agents,
thereby permitting an uncomplicated analysis of drug trans
port (20, 21). However, cyclophosphamide, with an intact ring
structure, is inactive as an alkylating agent (12, 16, 42), thus
providing an ideal opportunity for studying drug transport in a
pure form.
substrate
was not identified for the cyclophosphamide
carrier, and
transport was by a mechanism separate from that of other
alkylating
Previous studies have demonstrated that the alkylating agent
HN23 is transported by an active, carrier-mediated process in
murine L5 178Y lymphoblasts (20, 21) in normal and leukemic
human lymphoid cells (34), and in rat Walker 256 carcinosar
Institute
of
MATERIALS AND METHODS
Cell Cultures. Murine leukemia L5 l78Y lymphoblasts with
a doubling time of 10 to I I hr were grown in cell culture as
previously described (18, 20, 21). Exponential phase cells
adjusted to a concentration of 2.5 to 3 X 106 cells/mI in
Fischer's medium (Grand Island Biological Co., Grand Island,
N. Y.) were used for all transport
studies.
Chick embryo liver cells were prepared in suspension by
pooling 4 to 6 livers from 15- to 19-day-old chick embryos,
digesting in 2.5% trypsin and 1% pangestin in Earle's balanced
salt solution at pH 7.0 at room temperature, and suspending
the cells by gentle agitation sequentially through a Pasteur
pipet, a 21-gauge and, finally, a 25-gauge needle. for 10 min
each, as described elsewhere (L. G. Israels, B. A. Schacter, B.
Yoda, and G. J. Goldenberg, submitted for publication).
Erythrocytes and cell clumps were separated from the liver
cells by gravity sedimentation of the cell suspension in 15-mI
3The abbreviations used are: HN2, nitrogen mustard; DNP,
dinitrophenol; CCCP, m-chlorophenyl carbonylcyanide hydrazone;
NEM, N-ethylmaleimide; P0MB, p-hydroxymercuribenzoate.
CANCER RESEARCH VOL. 34
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Mechanism ofCyclophosphamide
centrifuge tubes for 3 to 5 min. The supernatant was
centrifuged at 2500 rpm for 10 mm (International centrifuge,
Universal Model LW), and the cell pellet consisting mainly of
liver cells was resuspended in Fischer's medium supplemented
with 10% horse serum. The cell suspension was allowed to
stand at room temperature for 20 mm; further removal of
contaminating RBC was achieved by low-speed centrifugation
Transport
velocities, S is substrate or drug concentration, and K1 and K2
are the Michaelis constants for each of the 2 uptake
components, as described by others (7, 8, 39—41).
The values for V, , K, , V2 , and K2 , which are kinetic
parameters, corrected not only for binding but also for
2-component
interaction,
were calculated
from the following
formulas derived by Neal (37):
at 100rpmfor5 mm.Clumpsof livercellsweredispersed
by
further enzyme treatment with trypsin and pangestin for 10 to
I 5 mm at room temperature. The final liver cell suspension in
Fischer's medium gave a homogenous size-distribution plot in
lrM,
K1,K2=—I—+
2[J,
M1—M2
±
‘2'1
the CoulterModelB electronicparticlecounter,andcell
viability was at least 90% as determined by trypan blue dye
exclusion. Cell size was determined in the Coulter counter
calibrated with giant ragweed pollen (mean cell diameter, 19.5
i.tm) and paper mulberry spores (mean cell diameter, 12.5 pm),
both of which were obtained from Coulter Diagnostics, Inc.,
Miami Springs,
Fla. The cell volume
(mean
K,
V, =(T
±S.E.) of chick
Transport
5@14C
Studies.
described
Transport
studies
were performed
by
previously (20, 21). Cyclophosphamide
monohydrate
(specific
activity
ranging
from
4 to
9.9
mCi/mmole) and 3-O-methyl-D-glucose-' 4C (specific activity,
10 mCi/mmole) were obtained from New England Nuclear,
Boston, Mass. Cyclophosphamide-' 4C monohydrate
was
diluted with appropriate amounts of unlabeled cyclophos
phamide monohydrate (Mead Johnson, Evansville, md.) to
give specific
activities
ranging
from
—
M2
.—
\M1
@i,M1—M2\2
121,JI
Ivi7A@@@
embryo liver cells was 1259 ±I 7 cu pm, which was similar to
that of L5 178Y lymphoblasts (1 273 ±28 cu pm) reported
previously (19).
methods
1 —41
I I—+
‘2_I,M2J@
M1—M2
1' I
‘2'1 .)/(K1
K2)
V2 = l/I,—V1,
where M, and M2 are the observed slopes and I@ and ‘2are
the observed intercepts for each of the 2 components of drug
transport.
All data were analyzed by a 2-tailed t test comparing the
significance
of the difference
of the means.
RESULTS
0.6 to 3.4 mCi/mmole.
Activated cyclophosphamide was prepared by the method of
Connors et a!. (12), using a NADPH-generating system and
mouse hepatic microsomes.
Incubations were terminated by rapid chilling to 4°and
centrifugation through a layer of 0.25 M sucrose in Hopkins
vaccine tubes to remove extracellular radioactivity . The cells
were solubiized in 0.5 N NaOH, and radioactivity was
determined by liquid scintillation spectrophotometry
using
Aquasol (New England Nuclear). Radioactivity in the cells was
compared to that in an equivalent volume of medium, the
result being expressed as cell/medium ratio.
Time Course
of Cydophosphamide
Uptake.
A time course
of the uptake of 1 mM cyclophosphamide-' 4C by L5 I 78Y
lymphoblasts in vitro is shown in Chart 1. After rapid binding,
uptake
was approximately
linear for 20 mifl and then entered
a plateau phase that approached a cell/medium ratio of unity.
Evidence that the drug was transported into the cell was
that the percentage of total cell radioactivity found in the cell
sap fraction was 89.6 ±4.7%, and that in the membrane
fraction was 1.3 1 ±0.7%, for cells exposed to 0.5 mM
cyclophosphamide for 10 mm at 37°.
Kinetic Analysis of Cyclophosphamide Uptake. The rela
At time points up to 1 mm, a cell/medium ratio of 0.14 ± tionship between cell/medium ratio and cyclophosphamide
concentration is illustrated in Chart 2. The concentration
0.02 was observed at cyclophosphamide concentrations rang
gradients decreased as drug concentration increased from 0.25
ing from 0.1 to 10 mM , as illustrated in a typical time course
to 1 mM , but thereafter as drug concentration increased, no
of drug uptake (Chart 1). This value of 0.14, which
presumably represents rapid drug binding to the cell mem
brane as noted by others (9, 11, 22, 32), was subtracted
routinely from the observed cell/medium ratio at subsequent
times, in order to measure
@
carrier-mediated
transport.
Uptake
was also expressed as velocity of drug uptake in moles/min/
cell, which likewise was corrected for rapid binding.
The kinetics of drug uptake, in this study, may be described
as the sum of 2 Michaelis-Menten equations with different
kinetic parameters;
v=
V1.S
K1+S
+
V2.S
Q 0.8
a
0.4
U
0
60
120
180
TIME (MINUTES)
K2+S
where v is total velocity and V, and V2 are the maximum
Chart 1. Time course of the uptake of 1 mM
am4
C
by L5 178Y cells at 37°
. The uncorrected uptake, expressed as
cell/medium ratio, is plotted against time.
DECEMBER 1974
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3275
G. I. Goldenberg et aL
Table 1
The Km and Vm@ for low- and high.dose cyclophosphamide transport
by L51 78Y lymphoblasts in vitro.
Cyclophosphamide
concentration
(mM)Km
mole/mm/cell)Low
@
(mM)Vm@
(X 10 ‘
dose, 0.25—10.39
±0.03a0.49
0.07@Highdose,l—1075±2949±14
a Mean
(CYCLOPIIOSPHAMIDE
@
@C)mM)]
Chart 2. Uptake of cyclophosphamide-' C by L51 78Y cells with
cell/medium ratio corrected for rapid binding plotted as a function of
drug concentration. L5178Y lymphoblasts at a concentration of 2 to 3
± S.E.
of
6 determinations
obtained
±
by
linear
regression
analysis of Lineweaver-Burk plots, after correction for binding and
application of the Neal analysis for 2-component transport, as described
in the text. A 2-tailed t test showed that the differences of the mean
Km and mean Vmax were highly significant (p < 0.00 1).
x 10'cells/ml
were
incubated
for10minat37°
atdrug
concentrationsThe same data were plotted according to the method of
ranging from 0.25 to 10 mM.
further drop in gradient was noted. The decrease in cellular
penetration with increasing substrate concentration is due
presumably to saturation of carrier sites and is characteristic of
a carrier-mediated process (2).
Cyclophosphamide uptake at a concentration range of 0.25
to 10 mM is presented as a Lineweaver-Burk plot in Chart 3.
The biphasic nature of cyclophosphamide uptake over this
40-fold
range
in drug concentration
represented
a deviation
from simple Michaelis-Menten kinetics, and suggested that
uptake consisted of 2 components. Evidence for saturation of
the low-dose component was the finding of a positive intercept
on the y axis. The high concentration component revealed an
intercept approaching the origin, but which nevertheless had
measurable kinetic parameters (Table 1).
Eadie and Augustinsson, which demonstrated
more dra
matically the biphasic nature of cyclophosphamide uptake
(Chart 4).
Resolution of Uptake Data into 2 Transport Systems.
Application of the Neal analysis for 2-component transport
(37), which was described in “Materialsand Methods― (see
above), established that the high-dose component was not
altered
appreciably
by the Neal correction,
but the observed
uptake overestimated the kinetic parameters for the low-dose
system (Chart 5).
The kinetic parameters obtained by application of the Neal
analysis for 2-component transport, to uptake data corrected
for binding, are shown in Table 1. Transport at low
8
3
6
>
>2
4
2
0
1
2
3
00.5
4
Chart 3. Lineweaver-Burk plot of cyclophosphamide
@
uptake by
L5178Y lymphoblasts corrected for rapid binding. Reciprocal uptake
velocity in moles/mm/cell X 10 ‘
â€ĩs plotted on the ordinate against
reciprocal mM drug concentration on the abscissa. The 2 lines were
obtained by linear regression analysis, and each component included
uptake at a drug concentration of 1 mM. The data represent the mean
of 6 determinations and the confidence intervals shown are the S.E.
S.E.'s are not shown where they are too small to be illustrated. The
linear regression equation of low-dose uptake wasy 0.5380x + 0.7592
with a correlation coefficient of 0.9716, and that for high-doseuptake
wasy = 1.2882x + 0.0188 with a correlation coefficient of 0.9976.
3276
1.0
1.5
v/s
1/[CYCLOPHOSPHAMIDEMC(mM)J
Chart 4. Eadie-Augustinsson plot of the same uptake data shown in
Chart 3. Uptake velocity in moles/rain/cell X 10 â€ĩs plotted on the
ordinate against the ratio of uptake velocity/cyclophosphamide
concentration (mM) on the abscissa. The 2 lines were drawn by linear
regression analysis. Both components included uptake data at a drug
concentration of 1 mM. The data represent the mean ±S.E. of 6
determinations. S.E.'s are not shown where they are too small to be
illustrated. The linear regression equation of low-dose uptake was y
—0.5635x+ 1.2798 with a correlation coefficient of —0.81
15, and that
for high-dose uptake was y —47.2219x+ 41.784 with a correlation
aefficient of —0.4678.
CANCER RESEARCH VOL. 34
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Mechanism ofCyclophosphamide
The Effect of Sodium on Cyclophosphamide Transport. The
effect of Na@ concentration on cyclophosphamide transport
was evaluated by comparing uptake at 10 mm in Hanks'
balanced salt solution containing 145 mEq Na@per liter and in
4
Hanks'
balanced
salt solution
2
±10.0% of the control,
0
1
2
3
1/[CYCLOPHOSPHAMIDE
4
14C(mM)J
Chart 5. Neal correction for 2-component transport of cyclophos
phamide by L5178Y lymphoblasts in vitro. The observed uptake plot
labeled
as
the
combined
plot,
has
been
resolved
into
separate
high
and low-dose uptake components.
@
cyclophosphamide concentrations appeared to be mediated by
a high-affinity, low-capacity system while, at high drug levels,
a low-affinity, high-capacity system predominated. The low
dose system had a K@ of 0.39 ±0.03 mM , which was within
the range of cyclophosphamide concentrations studied. How
ever, the high-dose component had a Km value of 75 ±29 mM,
well above the highest cyclophosphamide
concentration
examined. On the basis of kinetic analysis alone, it is not
possible to determine whether this 2nd phase of drug uptake
was by simple diffusion or a technically nonsaturable,
low-affinity transport system.
Temperature Dependence of Cyclophosphamide Uptake.
Low-dose cyclophosphamide uptake at 37 increased for up to
2 hr following the initial rapid, binding phase, whereas at 4°
only the rapid component of drug uptake was noted (Chart 6).
High-dose drug uptake was also temperature sensitive; uptake
of 10 mM cyclophosphamide for 10 mm, at 4°was 33.9 ±
3.2% of the control, and the difference was highly significant
(p < 0.001). Conversely, rapid binding as measured by uptake
at 1 mm was temperature independent.
U
0.5
0
60
120
TIME (MINUTES)
6. Time course of the uptake
of 0.1 mM cyclophos
phamide-' 4C by L5178Y cells at 37°(o) and 4°(.). Uncorrected
@
NaCI in
and this change was also significant
(p
3.0% of the control, and the difference was statistically
significant (p < 0.05).
One mM ouabain had no significant effect on the uptake of
0.5 mM cyclophosphamide, in that uptake in the presence of
ouabain was 94.3 ±2.8% of the control.
Effect of Metabolic Inhibitors on Cyclophosphamide Trans
port. The effect of several metabolic
inhibitors
on cyclophos
phamide transport is shown in Table 2. Oligomycin (0.1 mM)
and 1 mM cyanide resulted in significant inhibition of drug
uptake;
all other inhibitors
Evaluation
phamide
of the
Transport.
had no effect.
Chemical
The effects
Specificity
of Cyclophos
of a wide variety
of natural
substrates and several structural analogs of cyclophosphamide,
including the alkylating agents chlorambucil, melphalan, HN2,
isophosphamide, and activated cyclophosphamide, were ex
amined
and found
to have no effect
on cyclophosphamide
transport (Table 3).
Table 2
The effect of several metabolic inhibitors on cyclop/zosphamide
transport by L51 78Y cells in vitro.
The effect of metabolic inhibitors, at the concentrations indicated,
on the transport of 0.5 mM cyclophosphamide-' C by L5l 78Y cells, at
370,
for
10
mm;
The
cells
were
preincubated
with
the
inhibitor
for
30
mm.InhibitorConcen
of
deter
minations%
39NSSodium
fluoride20494.9
3.0NSP0MB0.028105.6
5.3NSNEM0.00754103.0
>
Chart
replacing
4.0@'<0.001Sodium
±
cyanide1.0871.6
3.4<0.001Sodium
±
cyanide0.1486.2
4.9NSCAntimycin
±
4.2NSDNP1.0490.8±7.1NSCCCP0.14101.3±7.5NSlodoacetate5.04109.8
A0.1496.1
±
0
0
Tris
< 0.01).
Sodium deprivation had no effect on the rapid-binding
phase of high-dose 10 mM cyclophosphamide
uptake, as
measured by uptake at 1 mm. However, uptake of 10 mM
cyclophosphamide in Na@-poor medium at 10 mm was 64.4 ±
tration
control0pOligomycin0.1863.7
(mM)No.
@
with
isotonic proportions, leaving a residual Na@concentration of 5
mEq/l. Drug uptake was corrected for binding and expressed
as a percentage of control uptake. Uptake of 0.1 mM
cyclophosphamide in Nat-poor medium was 45 .6 ±3.8% of
the control, and the difference was highly significant (p <
0.001). Uptake of 0.5 mM drug in the same medium was 40.4
>
(0),
Transport
uptake is plotted as moles/cell X 10
the abscissa.
6 on the ordinate against time on
a Results
were
corrected
±
±
±
±0.8NS
for rapid
binding
and are expressed
as the
percentage of the mean cell/medium ratio obtained in the absence of
inhibitor. The data represent the mean of 4 or 8 determinations, and
were analyzed statistically by the 2-tailed t test.
b Mean ±S.E.
C NS,
not
significant.
DECEMBER 1974
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3277
G. J. Goldenberg et al.
Table 3
Tue effect ofseveral alkylating agents on cyclophosphamide transport
by L51 78Y cells in vitro.
0.40
Q @35
of
deter
minations%
controlbChlorambucil1
Alkylating agent―Concentration
(mM)No.
S
I
.03107.4
±Melphalan0.54104.2
6.0HN20.58100.8
7.0Isophosphamide0.54105.7
±
±
4.0Activated
±
0.541
cyclo
1.9phosphamide
a Chlorambudil,
melphalan,
and
@0.30
U
0.23
00.5 ±
HN2
were
hydrolyzed
in 0.1
[CYCLOPHOSPHAMIDE
‘@C(mM)J
N
Chart 7. Uptake of
sphaime-4 C by chick embryo liver
cells with cell/medium concentration gradients, corrected for rapid
filtered through a Millipore membrane, as described previously (21).
binding, plotted as a function of chug concentration. Liver cell
Isophosphamide was not subjected to alkaline hydrolysis. Cyclophos
phamide was activated by the method of Connors et al. (12). The suspensions, prepared by methods described in the text, were incubated
alkylating agents were added simultaneously with labeled cyclophos
at a concentration of 2.6 X 106 cells/mI, for 10 mm at 37°at drug
phamide, and the cell suspension was incubated for 10 mm at 37°. concentrations ranging from 0.25 to 10 mM.
NaOH at 60°for 2 hr, the pH was adjusted to 7.5, and the solution was
Cy4
C was used at a concentration of 0.5 mM,except
for the study with chlorambucil, in which case, 1 mM substrate was
used.
b Results
are expressed
as a percentage
of the control
2
cell/medium
ratio, as described in Table 2. All uptake values did not differ
significantly from control uptake as analyzed by the 2-tailed t test.
CMean±SE.
The amino acids cycloleucine, a-aminoisobutyric
acid,
proline, and phenylalanine ; the nucleic acid derivatives hypo
xanthine, uracil, adenine, 6-methyladenine, cytosine, thymine,
and adenosine; and the compounds, nicotine, nicotinamide,
and phenobarbital also had no effect on cyclophosphamide
transport. The last 3 compounds were tested because of
reports
@
that activation
of cyclophosphamide
was blocked
by
nicotine and nicotinamide (27, 42) and was stimulated by
phenobarbital (15, 43).
Cyclophosphamide uptake was determined in the presence
of 2 specific inhibitors of glucose transport (46) to determine
whether cyclophosphamide might be transported on a sugar
carrier. Uptake of 0.5 mM cyclophosphamide-' 4C in the
presence of 0.1 mM phloretin was reduced to 87.8 ±2.0% of
control uptake, and the difference was statistically significant
(p < 0.01); drug uptake in the presence of 1 mM phiorizin was
also reduced to 85.7 ±3.8% of the control, but the difference
fell short of statistical significance. For more direct examina
tion of the interaction of glucose and cyclophosphamide
transport, uptake of 0.05 mM 3-O-methylglucose-1 4C was
determined in the presence of 0.1 and 1 mM unlabeled
cyclophosphamide acting as potential inhibitor. Cyclophos
phamide did not inhibit uptake of the labeled sugar.
Cyclophosphamide
Uptake by Chick Embryo Liver Cells. A
time course of cyclophosphamide
uptake by chick embryo
liver cells at 37°and 4°indicated that, as with L5178Y cells,
uptake was temperature dependent and contained a rapid
binding component. A single kinetic analysis of drug uptake
by liver cells ifiustrated a decrease in uptake with increasing
drug
concentration
(Chart
7). A Lineweaver-Burk
plot
of
cyclophosphamide uptake was biphasic (Chart 8), with a Km
of 0.43 mM and a Vmax of 1.05 X 10@“
mole/mm/cell for
low-dose transport, after correction for binding and applica
tion of the Neal analysis for 2-component transport, findings
similar to those obtained with L5 178Y cells (Table 1).
3278
>
0
1
2
3
4
1/[CYCLOPHOSPHAMIDE‘@C
(mM)]
Chart 8. A single experiment illustrating the Lineweaver-Burk plot of
cyclophosphamide uptake by chick embryo liver cells, corrected for
rapid binding as described in the text. Reciprocal uptake velocity in
moles/mm/cell x 10
is plotted on the ordinate against reciprocal
mM drug concentration
on the abscissa.
The 2 lines were obtained
by
linear regression analysis, and each component included uptake at a
drug concentration of 1 mM. The kinetic parameters shown are for the
low-dose transport component.
Cyclophosphamide
characterized
transport
by an apparent
in the
12.4 X 10-i 7 mole/mm/cell,
those
demonstrated
high-dose
range was
Km of 6.9 mM , and a Vmax Of
values which were lower than
for high-dose
transport
by L5178Y
cells.
DISCUSSION
Binding of Cyclophosphamide
to the Cell Surface
The rapid binding of cyclophospharnide
to L5 178Y
lymphoblasts
was temperature
and sodium independent,
contrasting with subsequent slower transport, which was
carrier
mediated
and
temperature
Uptake
due to rapid binding
uptake
to ensure
that
and
sodium
was subtracted
initial uptake
dependent.
from the total
velocity
was being
measured.
Evidence that cyclophosphamide
traverses the cell mem
CANCER RESEARCH VOL. 34
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Mechanism ofCyclophospha,nide
brane
was
membrane
phamide
shown
by
the
distribution
and cell sap fractions.
appeared
in the
cell
of
drug
between
Most of the cyclophos
sap,
with
relatively
small
quantities in the membrane fraction. Furthermore, the small
amount of radioactivity in the membrane fraction indicated
that washing effectively removed the binding of drug to the
cell
surface
(11,22).
which
oubain
The uptake of cyclophosphamide
brane
Transport
across the plasma mem
of L5 178Y cells was mediated
by 2 distinct
processes.
This was revealed most clearly by the Eadie-Augustinsson plot
of drug uptake (Chart 4). The Lineweaver-Burk plot of
cyclophosphamide transport was resolved into 2 independent
components by the Neal analysis. This correction markedly
altered the calculated kinetic parameters of low-dose transport
but had little effect on the high-dose component (Chart 5).
Kinetic
analysis
of low-dose
uptake
showed
evidence
of
saturabiity. Absolute saturation could not be shown because
of superimposition of the high-dose component. The demon
stration that the rate of uptake of a substrate approaches a
limiting saturation value with increasing concentration is
strong evidence of carrier-mediated transport (45).
High-dose cyclophosphamide uptake was technically non
saturable,
so kinetic
analysis
could
not discriminate
or no effect
on Na@-dependent
transport (4, 14, 25, 35). Cyclophosphamide uptake appears
to be another example of Na@-dependent, ouabain-insensitive
transport.
Hillman
and Rosenberg
(30) suggested
that Na@may act at
more than 1 transport step, and that it may have different
mechanisms of action in the total transport process. They
examined
Kinetic Analysis of Cyclophosphamide
has had little
Transport
both the substrate-carrier
interaction
and membrane
translocation processes of proline uptake by renal tubules (29,
30). Interaction of proline with the carrier was sodium
dependent but ouabain insensitive, whereas transport across
the tubule membrane was both sodium and ouabain sensitive.
The partial N@ dependency and ouabain insensitivity of
cyclophosphamide transport may be explained by a sodium
requirement for the interaction of substrate and carrier.
Evidence
against
a
Na@ requirement
for
the
membrane
translocation of cyclophosphamide
was the finding that
uptake was not inhibited by ouabain, and did not proceed
against a concentration gradient, which would occur with
Na@-linked cotransport.
Sodium ions may act on the cyclophosphamide carrier,
producing allosteric changes at the active site. Bihler (2) has
speculated that Na@ produces an allosteric change in the
intestinal sugar carrier such that the affinity for certain sugars
is increased.
between
mediated transport and uptake by passive diffusion. However,
the demonstration that high-dose cyclophosphamide uptake
was temperature and sodium dependent suggested a carrier
Effect of Metabolic Inhibitors on Cyclophosphamide
mediated
rable, low-affinity, carrier-mediated systems have been de
scribed with Km values comparable to that obtained for
cyclophosphamide (2, 5, 6, 28). The existence of 2 systems for
transport of cyclophosphamide across the cell membrane is
not unusual, since there are many reports of more than 1
mode of mediated transport for a single organic molecule (4,
7, 8, 34, 39—41).
Metabolic inhibitors may interfere with the production or
utilization of energy, necessary for the function of the cell.
Facilitated diffusion transport systems, which do not accumu
late substrate against a concentration gradient, do not require
a large input of energy, unlike an active transport process.
However, energy is required to maintain the structural
integrity of the cell membrane, and a reduction in energy may
also inhibit an inactive carrier-mediated process (45, 46).
Cyanide may block respiration at many sites; however,
Temperature Dependence of Cyclophosphamide
cytochrome
mechanism.
Furthermore,
many technically
nonsatu
Uptake
chain,
The uptake of 0.1 and 10 mM cyclophosphamide was
temperature sensitive. Stein (45) and Christensen and Liang (6,
10) state that temperature dependence is strong evidence for
mediated
transport
and tends to exclude
simple diffusion
as a
mechanism of uptake.
Sodium-dependent,
Ouabain-insensitive
Transport
of Cyclo
phosphamide
hans
port
oxidase,
appears
Antimycin
the terminal
to be the
enzyme
most
in the respiratory
sensitive
A blocks the flow of electrons
enzyme
(26).
from cytochrome
b
to cytochrome a in mitochondrial respiration (33).
Cyclophosphamide uptake in the low-concentration range
was significantly inhibited by 1 mM sodium cyanide, was
slightly inhibited by 0.1 mM cyanide but not to a significant
degrees, and was unaffected by 0.1 mM antimycin A. Higher
concentrations of antimycin A were not used because of cell
toxicity. Relatively high cyanide concentrations ranging from
5 to 10 mM have been used to characterizeother transport
The significance of sodium dependence as a criterion of
mediated transport is illustrated by investigations that corre
late Nä'-dependence with carrier-mediated transport (36, 38).
The requirement for sodium provides evidence for carrier
mediated transport of cyclophosphamide by both the low- and
high-dose systems. However, cyclophosphamide transport was
not affected by ouabain, a drug which disrupts ion gradients
by inhibiting the Na@-K@ pump (17). Although ouabain
sensitivity and sodium dependence are usually coupled
phenomena (13), many other systems have been described in
systems
(28, 29, 35, 40). Metabolic
inhibition
of intact
cells
may require high cyanide concentrations because of poor
membrane permeability of this anionic inhibitor (26), whereas
antimycin,
(45).
This
a lipid-soluble
precludes
agent, penetrates
a quantitative
comparison
cells more readily
of the
effect
of
antimycin and cyanide on cyclophosphamide transport.
The metabolic inhibitors DNP and CCCP uncouple oxidative
phosphorylation from mitochondrial respiration without in
hibiting the respiratory process (33, 44). Oligomycin blocks
ATP formation in a different way, by inhibiting both oxidative
DECEMBER 1974
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3279
G. J. Goldenberg et a!.
phosphorylation and respiration (44).
Oligomycin at a concentration of 0.1 mM significantly
inhibited cyclophosphamide uptake; however, no inhibition
was observed with either 0.1 mM CCCP or 1 mM DNP. A
similar rate of cell penetration probably occurs for all 3
inhibitors, as they are lipid soluble. The preferential sensitivity
of cyclophosphamide transport to oligomycin is similar to that
reported for other transport systems (14, 24).
Sulfhydryl groups do not appear to play a role in
cyclophosphamide transport since uptake was not affected by
the sulfhydryl reactive reagents NEM, P0MB, or iodoacetate.
The inability to inhibit drug transport with iodoacetate or
sodium fluoride suggests that glycolysis is not a significant
energy source for maintenance of cyclophosphamide trans
port.
Stein (45) lists the reduction of the rate of substrate
penetration by chemical inhibitors as a strong criterion for
facilitated diffusion.
Investigation of the
phamide Transport
Chemical
Specificity
of Cyclophos
General
Alkylating Agents. In an evaluation of chemical specificity,
other alkylating agents were tested for their effect on
cyclophosphamide uptake by L5 l78Y cells. The presence of
unlabeled cyclophosphamide inhibited the uptake of cyclo
phosphamide-' 4C. However, activated cyclophosphamide, iso
phosphamide, and hydrolyzed derivatives of chlorambucil,
melphalan, and HN2, were all ineffective in blocking transport
(Table 3). This suggests that cyclophospham.ide transport has a
narrow range in chemical specificity, since neither activated
drug nor isophosphamide, a close structural analog, had any
effect on drug uptake. Isophosphamide differs from the parent
compound only in that 1 chloroethyl group is attached to the
ring
activated cyclophosphamide,
melphalan, and chiorambucil
were transported by mechanisms independent of that de
scribed for HN2 (21). The independence of cyclophosphamide
and HN2 transport, together with the inability of other
alkylating agents to inhibit transport of either drug, suggests
that several mechanisms exist for transport of alkylating
agents.
Natural Substrates. Several naturally occurring substrates
were investigated in an attempt to identify a native substrate
for the cyclophosphamide carrier. However a wide range of
amino acids and several components of nucleic acids had no
effect on cyclophosphamide transport.
Despite the slight inhibition of cyclophosphamide transport
by phloretin and phiorizin, it is unlikely that the drug enters
L5 178Y cells on a sugar carrier, since unlabeled cyclophos
phamide had no effect on 3-O-methylglucose-' 4C transport.
Furthermore,
phiorizin and phloretin, although generally
considered as specific inhibitors of gluocse transport (46), have
been reported to inhibit transport of substrates other than
sugars (1, 31).
nitrogen:
Cl—CH3—CH2
\II/
/
0
N—P
Cl—CH2---CH2
N—CH2
\
\
/
CH2
Considerations.
amino
Cl
4H2
CH2
@/_C@2
N—P
H
CH2
\
Liang
(9)
em
acid, diethyiglycine,
prompted
Christensen
and Liang
GoodandRose(23) suggested
thatcompounds
thatappear
to have radically different structural characteristics may adopt
similar conformations in solution, due to structural changes
related to hydration. Chemical specificity may relate to
allosteric conformation
in solution (23), and transport
specificity may include chemical recognition of nonbiological
molecules (9, 10). Cyclophosphamide may utilize a transport
system with specificity toward similar heterocycic compounds
or, alternatively, the drug may exhibit structural conformation
in solution resembling a metabolite that has not been
examined in this study.
O—CH2
Cyclophosphamide
7
and
(9) to speculate that there may be other transport systems for
nonbiological substances.
Cyclophosphamide
Cl—CH2—C@2
Christensen
phasized that failure of a particular substance to inhibit known
transport systems does not exclude a carrier-mediated trans
port mechanism for that substrate. The demonstration of a
transport system functioning predominantly for the synthetic
Transport by Chick Embryo Liver Cells
The demonstration of heterogeneous kinetics and tempera
ture dependence suggests that transport of cyclophosphamide
by chick embryo liver cells resembles that noted in L5178Y
lymphoblasts, both being carrier mediated. Since the liver is
the principal site of cyclophosphamide activation (3, 16, 27,
42), the demonstration of carrier-mediated transport of intact
drug by liver cells adds considerable relevance to this report
and sheds further light on the basic pharmacology of
cyclophosphamide.
/
O—CH2
ACKNOWLEDGMENTS
Isophosphamide
The lack of inhibition by other alkylating agents suggests
that transport of cyclophosphamide is by an independent
mechanism. Previous work showed that both intact and
3280
We thank J. M. Anderson and J. A. Lepp for their excellent technical
assistance, Dr. W. A. Zygmunt of Mead Johnson Research Center,
Evansville, md., for supplying unlabeled cy'clophosphamide mono
hydrate,
and Dorothy
Faulkner
for typing the manuscript.
CANCER RESEARCH VOL. 34
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Mechanism ofCyclophosphamide
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CANCER
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VOL. 34
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Mechanism of Cyclophosphamide Transport by L5178Y
Lymphoblasts in Vitro
Gerald J. Goldenberg, H. Bernard Land and Douglas V. Cormack
Cancer Res 1974;34:3274-3282.
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