[CANCER RESEARCH 34, 371-377, February 1974]
Stereochemical Characteristics of the Folate-Antifolate
Mechanism in L1210 Leukemia Cells1
Transport
Francis M. Sirotnak and Ruth C. Donsbach
Memorial Sloan-Kellering Cancer Center, New York. New York 10021
SUMMARY
The rate of influx, extent of concentrative uptake, and the
rate of efflux (loss) by active transport in L1210 leukemia
cells has been compared for the pteridine antifolates, aminopterin and methotrexate, eight related quinazoline analogs,
and two pyrimidine derivatives. The data reveal a difference
in the Stereochemical specificity for influx and efflux. Influx
is preferential in the order pteridine, quinazoline, and py
rimidine. Influx of aminopterin is more rapid than that of
methotrexate. L-Glutamylquinazolines were taken up faster
than L-aspartylquinazolines, but influx of a D-glutamylquinazoline was slower than the corresponding D-aspartyl
derivative. Influx of the quinazolines was faster when there
was a methyl- or chloro- substitution at position 5. Influx of
the pyrimidines was also faster when a methyl group was at
position 6. Michaelis constants (Km) for influx of the various
analogs varied from 1.42 x 10~6 Mto over 10"4 M. Individ
ual Vmax values were essentially the same (1.87 to 2.22
nmoles/min/g dry weight). The relationship between the
values for initial velocity of influx (v), the Km and Vmaxob
tained with each analog are in agreement with that predicted
by the Michaelis-Menten equation and is consistent with the
notion that differences in rates of influx are attributable to
differences in the affinity of the carrier for the system. Ef
flux is preferential in the order pteridine, pyrimidine, and
quinazoline. Efflux of aminopterin and methotrexate occurs
at the same rate. Both aspartyl- and glutamylquinazolines
efflux at about the same rate, but the D-aspartyl and Dglutamyl forms efflux more rapidly than the corresponding
L forms. A methyl, and particularly a chloro, substitution
at position 5 of the quinazoline reduces the rate of efflux.
The extent of concentrative uptake observed for each analog
directly reflects the relative magnitude at which the influx
and efflux processes operate and may be the physiological
parameter most relevant to therapeutic efficacy.
INTRODUCTION
Recent findings (24, 25) from this laboratory attribute the
selective activity of methotrexate during therapy of the
L1210 leukemia to a greater persistence of drug in tumor
' This work supported in part by Grant CA-08748 from the National
Cancer Institute and Grant BC-108 from the American Cancer Society.
Received June 20, 1973; accepted November 7, 1973.
FEBRUARY
versus normal cells. This apparently occurs because of the
larger potential for concentrative uptake of drug by the
tumor cells after the serum concentration has fallen to a
low level. Although the significance of these findings to anti
folate treatment of human leukemia remains to be deter
mined, the potential for therapeutic exploitation of this
physiological site seems obvious.
The manner by which antifolates penetrate tumor cells
has been of interest to a number of workers (3, 5, 6-11, 1517, 20, 21, 23, 27, 28). There is extensive evidence (6-9, 16,
17, 20, 21, 23, 27, 28) in vitro indicating that uptake in
L1210 cells occurs by active transport. In most other tumor
cells, uptake has been shown (3, 5, 17) to at least resemble
an active transport process.
The characteristics of uptake in L1210 cells demonstrated
in vitro closely approximate that seen in the animal (24, 25).
In another aspect of these studies, the Stereochemical re
quirements of the transport mechanism in L1210 cells were
examined. Measurements were made of the rate of influx
and extent of concentrative uptake, as well as the rate of
efflux (loss) of a variety of folate analogs. The results of pre
liminary studies comparing aminopterin, methotrexate, and
methasquin have been reported from our laboratory (23).
Comparisons between methotrexate and methasquin (20)
and methotrexate and a pteroate analog (16) have also been
made elsewhere. A more extensive kinetic analysis involving
a number of individual Stereochemical differences among
analogs is presented here.
MATERIALS
AND METHODS
The maintenance and transplantation of the ascitic L1210
line (V) in vivo has been described (14). Methotrexate and
aminopterin were supplied by Lederle Laboratories, Pearl
River, N. Y. The quinazoline and pyrimidine analogs were
provided by Parke Davis and Co., Detroit, Mich. Amino
pterin and methotrexate were purified by chromatography
(22). The final purity of all drug samples was evaluated
bioautographically (4). The dihydrofolate reducÃ-asecon
tent of the LI 210 cells was determined by titration inhibition
with methotrexate or methasquin (30).
Enzyme Assay for Antifolate. The content of drug in cellfree extracts was determined by titration (30) with a par
tially purified (29) dihydrofolate reducÃ-asefrom a highlevel recombinant strain of Diplococcus pneumoniae (26).
The details of the routine tube assay have already been
1974
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371
Francis M. Sirotnak and Ruth C. Donsbach
described (23). All but 2 of the antifolates (see legend of
Table 1) titrate the microbial enzyme to about 80% inhibi
tion. Both D-deazaaminopterin and D-quinaspar titrate the
enzyme only to about 60% inhibition. However, in our ex
perience a good level of reliability in assays for both com
pounds can still be obtained by using additional sample
replicates. The range for the amount of drug detectable in
this assay varies between 0.05 and 0.5 ng.
Antifolate Uptake by I 1210 Cells. The harvesting of cells
has been described (23, 27). Usually 2 x IO7cells in 1 ml
(A6oo = 3.0) of suspending medium (pH 7.5) were incu
bated with drug. The uptake of drug at 37°corrected for
drug associating with cells at 0°was used as a measure of
uptake by active transport. Uptake at 0°(about 1 to 2% of
the total uptake at 37°)provides a measure of drug rapidly
adsorbed on the cell surface. This is essentially a tempera
ture-independent process (9, 23). In agreement with the
known lipophobic character of the folate analogs, the rate
of passive diffusion occurring within the concentration range
(2.2 n\i or less) used during these studies would be ex
pected to be negligible (2, 18). This was confirmed (6) for
methotrexate by an estimation made at 37°at an external
concentration (100 ßM)
well in excess of that necessary to
saturate the carrier mechanism. Similar measurements of
the diffusion at 37°of methotrexate were also made in the
current study at an external concentration of 500 ¿IM.
The
rate of diffusion at 0.45 JÕM
(the concentration used in the
rate determinations shown in Table 1) calculated from
Table 1
The uptake of folate antagonists by LI210 leukemia cells
Substituents
of
influx'
(nmole/min/
ring
acid
structure"2,4-Diami-nopteridine2,4-Diaminop-teridine2.4-Diamino-quinazoline2,4-Diamino-quinazoline2,4-Diamino-quanazoline2,
Compound"AminopterinMethotrexateD-Deaza-aminopterinDeazaaminopterin5-Methyldeaza-aminopterin5-Chlorodeazaami-nopterinD-QuinasparQuinasparMeth
M)1.424.9557.206.655.952.777.6538.3027.202
10"
5
6
moietyL-GlutamylCH,
10
gwt)0.7100.2300.0410.2080.2370.3500.1750.0470.06
dry
L-GlutamylD-GlutamylL-GlutamylCH3
L-GlutamylCl
i.-GlutamylD-AspartylL-AspartylCH3
L-AspartylCl
L-Aspartyl*
110180NSC
110191Basic
L-Aspartyl•
CHj
L-AspartylRate
" Aminopterin, /V-|p-|((2,4-diamino-6-pteridinyl)methyl]amino|benzoyl|-L-glutamate;
methotrexate. A'-(p-|((2,4-diamino6-pteridinyl)methyl]methylamino|benzoyl|-L-glutamate;
D-deazaaminopterin,
Ar-|p-|[(2,4-diamino-6-quinazolinyl)methyl]amino|benzoyl|-i)-glutamate,
hemihydrate; deazaaminopterin, N-\p |[(2,4-diamino-6-quinazolinyl)methyl]amino|benzoyl](- Lglutamate: 5-methyldeazaaminopterin, W-|/>-|[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino|benzoyl|-L-glutamate,
disodium, tetrahydrate; 5-chlorodeazaaminopterin, Ar-(/j-|[(2,4-diamino-5-chloro-6-quinazolinyl)methyl]amino|benzoyl|-L-glutamate,
hemihydrate; D-quinaspar, /V-(p-{[(2,4-diamino-6-quinazolinyl)methyl]amino|benzoyl|-D-aspartate,
disodium, heptahydrate;
quinaspar, /V-|/7-|[(2,4-diamino-6-quinazolinyl)methyl]amino|benzoyl|-L-aspartate;
methasquin, /V-(/>-|((2,4-diamino-5-methyl6-quinazolinyl)methyl]amino|genzoyl|-L-aspartate. disodium, pentahydrate; chlorasquin, /V-|p-|[(2,4-diamino-5-chloro-6-quinazolinyl)methyl]amino|benzoyl|-L-aspartate, dihydrate; NSC 110180, Ar-(p-|(/)-(2,4-diamino-5-pyrimidinyl)benzoyl]amino|benzoyl|-L-aspartate; NSC 110191,/V-jp-|[p-(2,4-diamino-6-methyl-5-pyrimidinyl)benzoyl]amino|benzoyl|-L-aspartate.
"p-Aminobenzoyl moiety attached at position 9 of the pteridinyl ring and benzylaminobenzoyl attached at position 5 of the
pyrimidines.
' The initial rate of uptake at 37°corrected for uptake at 0°.Rate = nmoles/min/g dry weight (drug],,«,!,.]= 0.45 M. Values
are an average of 4 to 6 replicate experiments, with a standard deviation of less than 30%. Each compound was always compared to
methotrexate run as an internal control in the same experiment.
" Michaelis constant (molar). Values are an average of 5 to 8 replicate experiments, with a standard deviation of less than 30%.
Each compound was always compared to methotrexate run as an internal control in the same experiment. Precautions used in deter
mining rates for true initial velocity were the same as described previously (6, 15).
' Not applicable, since this is a 5-arylpyrimidine (see above).
372
CANCER RESEARCH
VOL. 34
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Antifolate Transport in L/210 Leukemia Cells
these data was 0.00075 to 0.001 nmole/min/g dry weight.
The suspending medium consisted of 107 HIMNaCl, 5.3
mM KC1, 26.2 mM NaHCO3, 1.9 mM CaCl2, 1 mM
MgCl2-6H2O, 10 mM glucose, and 10 mM Tris-HCl. Ex
posure to drug was terminated by the addition of 10 ml
of cold 0.02 M potassium phosphate-0.14 M NaCl, pH 7.5.
Cells were washed free of drug by resuspension twice in the
same solution to a final volume of 1 ml. The cell number
was determined by an absorbance measurement using a
standard curve relating microscopic cell count to absorb
ance (27). Drug was removed from the cells by heat extrac
tion, and cellular debris was discarded after centrifugation.
Kinetic Analysis of Antifolate Influx. The Michaelis con
stant (Km) for the transport system was determined by the
method of Lineweaver and Burk (19). In this system, it
represents the external drug concentration needed for halfsaturation and is used as a relative measure of the affinity of
the carrier component for drug. The initial rate of influx at
37°(minus uptake at 0°)was measured at varying concen
trations of each antifolate. The time of incubation was ad
justed to allow for measurement of uptake below the dihydrofolate reducÃ-asecontent. Since no free drug exists
internally in this range, uptake is essentially unidirectional
and a valid measure of influx. The necessity for this pre
caution has been stressed in earlier reports (9, 23).
Measurements of Efflux by L1210 Cells. The efflux of the
various antifolates from LI210 cells was measured (9, 23)
by preloading the cells with drug (usually to an intracellular
level 3 to 4 times the dihydrofolate reducÃ-asecontent) and
washing the cells free of drug with cold 0.02 M potassium
phosphate-0.14 M NaCl, pH 7.5. The cells were then resuspended in fresh medium and incubated at 37°for vary
ing periods of time.
RESULTS
The initial rate of temperature-dependent influx of the
various analogs, at a concentration of 0.45 ¿tM,
is shown in
Table 1. The relative rate of influx among the group varied
more than 200-fold. Influx of aminopterin was the most
rapid, while influx of the pyrimidine analogs, NSC 110180
and NSC 110191 was the least rapid. The rate of influx of
methotrexate, and the related L-glutamylquinazoline, deazaaminopterin, was only one-third to one-fourth as rapid
as that of aminopterin. The L-aspartylquinazoline analogs
were transported at one-fifth the rate of the corresponding
L-glutamylquinazolines. 5-Chlorodeazaaminopterin and the
5-chloro-L-aspartylquinazoline (chlorasquin) were trans
ported at a rate almost twice that of the corresponding unsubstituted derivatives. A somewhat smaller increase in the
rate of influx occurred with 5-methyl-deazaaminopterin and
methasquin. The 6-methyl 2,4-diaminopyrimidine analog
(NSC 110191) had a higher rate of influx when compared
to the unsubstituted 2,4-diaminopyrimidine (NSC 110180).
The influx of the D-glutamylquinazoline, D-deazaaminopterin, occurred at only one-fifth the rate obtained with
L-deazaaminopterin. In contrast, the rate of influx of the
D-aspartylquinazoline (o-quinaspar) was 4-fold greater
than that of the corresponding i.-aspartyl form (quinaspar).
Kinetic Analysis of Antifolate Influx. Saturation (Michaelis-Menten) kinetics for temperature-dependent uptake
was demonstrated for all of the analogs except the 2 pyrimi
dine analogs (NSC 110180 and NSC 110191). The apparent
Michaelis constant (Km) derived for each analog is shown in
Table 1. As anticipated from the data on the rate of influx,
the transport mechanism appears to exhibit the greatest
affinity for aminopterin (Km = 1.42 x 10"6 M) and some
what less for methotrexate (Km = 4.95 x 10"6 M) and the
L-glutamylquinazolines (Km = 2.77 to 6.65 x 10~8M). The
mechanism seems to have even less affinity for the Daspartylquinazoline (Km = 7.6 x 10~6 M) and the Laspartylquinazolines (Km = 23.5 to 38.3 x 10"6 M) and
least for the D-glutamylquinazoline (Km = 57.2 x 10~6 M).
No Km value could be obtained with the 2 pyrimidine ana
logs because of the extremely high concentrations appar
ently necessary to saturate the system. The maximum rate
of influx obtainable (Vmax)with the pteridine and quanazoline analogs was calculated graphically or from the basic
Michaelis-Menten equation, v = (Vmax-S)/(K.m + S), where
v is initial velocity of influx and S is [drug]externai-Values
for each analog are approximately the same, varying from
1.87 to 2.22 nmoles/min/g dry weight.
The Intracellular Concentration of Antifolate at Steady
of the rate of influx of some of these drugs (sometimes by
kinetic analysis) by the natural folates folie acid, 5-methyl- State. The kinetics and extent of concentrative uptake for
tetrahydrofolic acid, and 5-formyltetrahydrofolic acid (7-9, each folate analog were measured by incubating L1210 cells
17, 21, 23, 27). A similar measurement of the inhibition of at 37°with drug (0.45 or 2.2 fiM)until the free intracellular
influx of the pyrimidine analogs at 37°by normal folates drug concentration was at equilibrium (steady state). This
was also made during the current studies. Like prior results, usually requires a period of 40 to 60 min. The data shown
inhibition of the rate of influx of these analogs was shown to in Table 2 and Charts 1 and 2 were corrected for uptake at
be competitive or resemble a competitive process and de 0°.This value is usually very small and has been shown (9,
monstrable only at saturating or near-saturating external 23) to represent mainly adsorption on the cell surface. The
concentrations. The extent to which the influx of each class kinetics of concentrative uptake of the various analogs
of antifolate was inhibited by normal folates was in good differed considerably. The accumulation of the pteridines
quantitative agreement with the relative rate and kinetics (Chart 1) is similar. Uptake occurs at essentially a constant
of influx observed for each.
rate (only influx occurs) until the dihydrofolate reducÃ-ase
The Influx of Antifolates by 1.1210 Cells. The folate ana
logs used during these studies include the pteridines, aminopterin and methotrexate, 8 quinazolines, and 2 pyrimidine
derivatives. In accordance with their gross stereochemical
similarity, the 3 groups appear to compete for the same
transport mechanism(s). This conclusion is based on a prior
demonstration of inhibition of the rate of influx at 37°of
methotrexate-3H by methasquin (20, 23) and of inhibition
FEBRUARY
1974
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373
Francis M. Sirotnak and Ruth C. Donsbach
Table 2
The concentrative uptake of folate antagonists by Li 210 leukemia cells
drugCompound"AminopterinMethotrexateD-DeazaaminopterinDeazaaminopterin5-Methyldeazaaminop-terin5-Chlorodeazaaminop-terinD-Quina
Intracellular
wt)21.915.912.931.244.841.651.380.135.522.935.340.32.21.6Freec(UM)11.127.465.6216.9
tern»5.063.392.5537.5911.4010.6366.5021.258.855.358.8010.18
110180NSC
110191(drugJe.uMT.ai6(MM)2.22.22.20.452.22.20.452.22.22.22.22.211.011.0Total(nmolesdry
°See Table 1 for structural details.
' External drug concentration.
' Based on a volume of 0.005 ml for the water content of the intracellular free space in 2 x IO7
cells (20). The amount of drug bound to dihydrofolate reducÃ-ase(3.75 nmoles/g dry weight) was
substracted from the total amount accumulated intracellularly. Values are based on 4 to 6 replicate
experiments with a standard deviation of less than 30%.
60r
40
[drug]ext
= 2.2 u M
dea/a .iminopterin
aminopterÃ-n
methotrexate
60
Chart I. The rate of concentrative uptake at 37°of pteridine and pyrimidine antifolates in LI210 leukemia cells. The uptake of each analog
was always related in 4 to 6 replicate experiments to the uptake of metho
trexate. Total uptake corrected for uptake at 0°.ext, external.
60
Chart 2. The rate of concentrative uptake at 37°of quinazoline anti
level is reached. The rate then gradually diminishes as the
steady-state level is approached. With the exception of
D-deazaaminopterin, quinaspar, and possibly D-quinaspar,
374
folates in L1210 leukemia cells. The uptake of each analog was always re
lated in 4 to 6 replicate experiments to the uptake of methotrexate. Total
uptake corrected for uptake at 0°.ext, external.
CANCER
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Antifolate Transport in L/2/0 Leukemia Cells
25 r
the kinetics of uptake for the quinazoline analogs appears
(Chart 2) to be quite different. Uptake of drug continues at
•¿
D -dea/a-aminopterin
o
deaza-aminopterin
a constant rate to internal concentrations far greater than
A
5-methyl, deaza- aminopterin
the drug equivalence of the dihydrofolate reducÃ-asecon
A
5-chloro. deaza aminopterin
tent. In these cases linear kinetics of uptake continue against
•¿
D-quinaspar
a sizable concentration gradient (see below). This is dra
O
quinaspar
matically apparent in the case of 5-chlorodeazaaminopterin
X
methasquin
-+•chlorasquin
where drug is accumulated to nearly 14-fold the dihydro
folate reducÃ-aselevel. At an exlernal concenlralion of 2.2
¿tM,the total accumulation of the pyrimidine analogs
(Chart 1) did not reach the dihydrofolate reducÃ-aselevel.
The extenl of concenlralive uplake observed for each folale analog varied over a wide range. The inlernal concenIralions of drug, al Ine sleady-slale levels shown in Charts
1 and 2, are given in Table 2. At an exlernal concentration of
2.2 /iM, uptake of ihe pleridine (Charl 1), aminoplerin,
and melholrexale was at the least concentrative (3- to 5fold higher internal concentralion). Uplake of all of the
quinazoline analogs (Chart 2), excepl D-deazaaminoplerin
and quinaspar, was considerably more concenlralive (al
leasl 8.8-fold higher inlernal concenlralion). 5-Chlorodeazaaminoplerin was concenlrated to Ihe grealesl exlent.
Al an exlernal concenlralion of 2.2 ¡J.M,
ihe inlernal level
al equilibrium was over 21 limes grealer. Al an external
concenlralion of 0.45 ¿tM,ihe difference was 70 times
60
grealer. Al ihe same exlernal concenlration (0.45 jtM),
t (min)
deazaaminoplerin was concenlraled almosl 40-fold. ConChart 4. The efflux of quinazoline antifolates by L12IO leukemia cells.
cenlrative uptake of the pyrimidine analogs, NSC 110180
and NSC 110191, could not be demonstraled al ihese con The data shown are based on the average of 3 to 5 replicate experiments in
which the efflux of methotrexate was measured as an internal control.
centrations.
The Efflux of Folate Analogs by L1210 Cells. Previous
studies (8, 9, 17, 23) have shown thai the loss of folate ana
logs from L1210 cells is temperalure dependent. This is in
20
agreemenl wilh ihe idea lhal the same carrier is ulilized for
bolh influx and efflux. A similar lemperature dependence
for efflux of all of the analogs has been observed during the
current study. The efflux of the various folate analogs from
L1210 cells in drug-free medium at 37°is shown in Charts
•¿
aminopterin
o methotrexate
3 and 4. Efflux of both the pteridine and pyrimidine analogs
A NSC 110. 180
(Chan
3) occurred very rapidly. The inlracellular drug level
A NSC 110. 191
was al ihe dihydrofolale reducÃ-aselevel within 10 to 15 min
after incubation was initiated. As reported earlier (8, 9, 23),
further loss of drug with lime occurred al an almosl imperceplible rale. Efflux of all of Ihe quinazoline analogs oc
curred (Charl 4) al a much slower rate. The internal level of
only D-deazaaminopterin, deazaaminopterin, and D-quin
aspar reached enzyme level during ihe 60-min efflux period.
Since Ihe decrease in concenlration of free drug in L1210
cells approximates a 1si-order process, il was possible more
accuralely lo quanlilale Ihe relalive rale of efflux for each
analog. A /i/2 value (lime required for a 50% decrease in
concenlralion) was first calculaled from a linear semilogarilhmic plol of ihe internal concentration of free drug at
various time inlervals. Values derived for each analog are
t (min)
given in Table 3. The rale conslanl for efflux for each is obChart 3. The efflux of pteridine and pyrimidine antifolates by L1210
lained from ihe expression k = In 2//i/2. These values are
leukemia cells. The data shown are based on the average of 3 to 5 replicate
also given in Table 3. A relalive difference in efflux rale of
experiments in which the efflux of methotrexate was measured as an inter
aboul 20-fold was demonslraled for Ihe various analogs.
nal control.
FEBRUARY
1974
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375
Francis M. Sirotnak and Ruth C. Donsbach
Table 3
The rale of efflux affatale antagonists by LI 210 leukemia cells
Experiments showing a rapid loss of aminopterin and
methotrexate suggest an interaction with the efflux mecha
nism which again is most efficient in the case of the pteridine
constant'
(min-1)0.2390.2020.0470.0340.0270.0110.0530.0190.0170.0180.1470.158
Compound"AminopterinMethotrexateD-DeazaaminopterinDeazaaminopterin5analogs. However, in contrast to that seen during influx, the
(min)2.93.414.520.526.263.813.136.240.739.84.54.4Rate
methyl group at A"°has little effect on efflux. Moreover,
both of the pyrimidine analogs efflux almost as rapidly as
the pteridines, suggesting a nearly equivalent affinity for the
same system. The quinazoline analogs as a group appear to
Methyldeazaaminopterin5-ChlorodeazaaminopterinD-QuinasparQuinasparMethasquinChlorasquinNSC
have the lowest affinity for the efflux mechanism. These are
lost from the L1210 cells slowly, with little difference ob
served between the L-aspartyl and L-glutamyl derivatives.
Both D-aspartyl and D-glutamyl analogs efflux more effi
ciently than the corresponding L forms. Whereas a methyl
10180NSC
1
or chloro substitution at position 5 of the quinazoline ring
110191l¿
was found to potentiate influx, the opposite is true for
efflux.
°Structural details are given in the legend of Table 1.
"Time required for internal concentration of free drug to decrease by
It is vividly apparent that both influx and efflux play a
one-half. Values are averages of 3 to 5 replicate experiments with a stand
significant role in determining the internal level of free drug
ard deviation of less than 30%.
achievable at a specific external concentration. Moreover,
' k = In 2/t*.
the kinetics and extent of concentrative uptake actually ob
served for each analog quantitatively reflects the magnitude
Aminopterin had the highest rate of efflux (k = 0.239), at which each can operate. For example, when both influx
followed by methotrexate and the 2 pyrimidine analogs, and efflux are relatively rapid (aminopterin) or slow (methNSC 110180 and NSC 110191. Efflux of D-deazaaminoasquin), fairly high intracellular levels are achievable at
pterin and D-quinaspar was slower, but at nearly the same
relatively low extracellular concentrations. When influx is
rate. The rate of efflux of the L-aspartylquinazoline analogs
slow and efflux is rapid, as in the case of the pyrimidine
was less than one-tenth the rate observed for aminopterin.
analogs (NSC 110180 and NSC 110191), only low intra
Two of the L-glutamylquinazolines, deazaaminopterin and
cellular levels are possible. On the other hand, when influx
5-methyldeazaaminopterin effluxed somewhat faster than
the corresponding L-aspartyl forms. The rate of efflux for is rapid but efflux is slow, phenomenally high intracellular
levels are possible, as with 5-chlorodeazaaminopterin.
the 5-chlorodeazaaminopterin was the lowest of the entire
Knowledge as to general therapeutic relevance of achiev
group (k = 0.011).
ing high steady-state levels of free drug in tumor cells will
require further study of these drugs at a pharmacological
DISCUSSION
level. As suggested (8, 23), the rate of influx alone is prob
The function of the antifolate transport mechanism in ably an inadequate parameter for evaluating possible ther
L1210 leukemia cells exhibits a considerable degree of di apeutic efficacy. This, in fact, has proved to be the case with
versity with respect to stereochemical specificity. Moreover, methasquin, which was relatively inert during experiments
the data reveal a difference in the stereochemical basis of measuring influx but is a fairly effective antileukemic agent
the influx and efflux processes. Based on the respective (12).
Although we have not directly demonstrated competition
apparent Michaelis constants for influx, the mechanism
seems to exhibit the greatest affinity for the 2,4-diamino
among various folate analogs for the efflux mechanism, it is
assumed that each analog utilizes the same carrier for this
analog (aminopterin) of folie acid. The affinity was some
what less in the case of the 2,4-amino, /V'-methyl analog purpose. This is probably true, if both influx and efflux oc
(methotrexate). The poor affinity of the mechanism for folie cur by a single carrier, since some evidence has been pro
acid itself has already been demonstrated (8, 9, 17, 23, 29). vided (Refs. 7 to 9, 17, 20, 21, 23, and 27, and in the present
On the other hand, 5-formyltetrahydrofolate (citrovorum study) which indicates that all of the analogs compete for
factor) and 5-methyltetrahydrofolate are probably trans
the same influx mechanism. Additional evidence for the in
ported by this mechanism to about the same extent as volvement of a single carrier for influx and efflux comes
methotrexate (8, 9, 17, 21, 23, 27). The mechanism has re from a demonstration of both countertransport (9, 21) and a
duced affinity for quinazoline derivatives and a severely transtimulation effect (7) in the L1210 system. The latter
reduced affinity for the corresponding pyrimidine analogs. was based on data showing that methotrexate influx is more
rapid in cells preloaded with 5-formyltetrahydrofolate. We
Interaction with the L-aspartyl analogs is poor in compari
son to the L-glutamyl analogs. The opposite seems to be have confirmed this finding and have also obtained the same
true with regard to analogs bearing D-glutamyl or D- result in connection with the influx of the quinazolines and
aspartyl moieties. The affinity of the mechanism is greater pyrimidines (F. M. Sirotnak and R. C. Donsbach, unpub
for quinazoline analogs bearing a methyl or especially a lished results).
chloro group at position 5. The system has greater affinity
The large spectrum of interaction between folate analogs
for a pyrimidine analog, if a methyl group is substituted at for the transport carrier is quite different from that seen
with the target enzyme, dihydrofolate reducÃ-ase.All of the
position 6.
376
CANCER
RESEARCH
VOL. 34
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Antifolate
analogs examined here are excellent inhibitors of the en
zyme in L1210 cells (Ref. 13; F. M. Sirotnak and R. C.
Donsbach, unpublished results) with dissociation constants
(K,) in the vicinity of 10~" M. The greater stereochemical
flexibility of the interaction with enzyme undoubtedly re
lates to the multisite nature (1) of the contact demonstrated
with this class of inhibitor. It would appear that related
studies (24, 25) in tumor versus normal cells, at the level of
membrane transport, might offer more opportunity for
elucidating exploitable stereochemical differences.
17.
18.
19.
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ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistance of Martha
A. Ward and the interest and support of Dr. Dorris J. Hutchison during
these studies.
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1974
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377
Stereochemical Characteristics of the Folate-Antifolate
Transport Mechanism in L1210 Leukemia Cells
Francis M. Sirotnak and Ruth C. Donsbach
Cancer Res 1974;34:371-377.
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