Download Antifolate Polyglutamylation and Competitive Drug Displacement at

[CANCER RESEARCH 46, 588-593,
February 1986]
Antifolate Polyglutamylation and Competitive Drug Displacement at
Dihydrofolate ReducÃ-aseas Important Elements in Leucovorin
Rescue in L1210 Cells1
Larry H. Matherly,2 Charles K. Barlowe, and I. David Goldman
Departments of Medicine and Pharmacology, Medical College of Virgin/a, Richmond, Virginia 23298
ABSTRACT
Previous studies from this laboratory have shown that the
addition of leucovorin to tumor cells dissociates methotrexate,
but not methotrexate polyglutamates, from dihydrofolate reduc
Ã-ase(L. H. Matherly, D. W. Fry, and I. D. Goldman, Cancer Res.,
43: 2694-2699, 1983). To further assess the importance of
these interactions to leucovorin rescue, antifolate growth inhibi
tion toward L1210 cells in the presence of leucovorin was
correlated with the metabolism of (6S)-5-formyl tetrahydrofolate
to dihydrofolate as a measure of dihydrofolate reducÃ-aseactivity.
Growth inhibition (greater than 95%) by methotrexate (5-10 UM)
following its intracellular polyglutamylation during a 3-h preexposure, or by continuous treatment with high levels of the
lipophilic antifolate, trimetrexate (1 /UM),was only slightly dimin
ished by 10 MMleucovorin (15-25%). High-pressure liquid Chro
matographie analyses of the derivatives formed from radiolabeled
(6S)-5-formyl tetrahydrofolate under these conditions showed an
incomplete conversion to dihydrofolate and metabolism to pre
dominantly 10-formyl tetrahydrofolate. Neither of the antifolates
interfered appreciably with the metabolism of the folate deriva
tives to polyglutamates. Growth inhibition in the presence of
leucovorin correlated with the accumulation of dihydrofolate
(1.5-2.2 nmol) from radiolabeled (6S)-5-formyl tetrahydrofolate,
reflecting continued suppression of dihydrofolate reducÃ-aseac
tivity at these drug concentrations. With lower equitoxic levels
of the trimetrexate (7.5 nM), the provision of leucovorin allowed
for a restoration of cell growth to a level greater than 90% of
control. Under these conditions, control levels of dihydrofolate
(0.2 nmol) were formed from radiolabeled cofactor, consistent
with sustained dihydrofolate reducÃ-aseactivity.
These findings support a role for the activation of dihydrofolate
reducÃ-ase as an important component of the reversal of the
effects of diaminoantifolates by leucovorin, presumably by a
competitive displacement of drug from the enzyme. Since no
displacement occurs in cells which have accumulated methotrex
ate polyglutamates, or in the presence of high levels of Irimetrexate, il appears that the concenlralion of unbound drug wilhin
cells is a significanl determinant of the extent of this competitive
binding interaclion. From these considerations, Ihe high levels of
methotrexate polyglutamates thai accumulate in sensitive tu
mors relative lo bone marrow and gaslrointestinal cells would
Received 6/27/85; revised 10/9/85; accepted 10/11/85.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' Supported by Research Grant CA-16906 and Training Grant CA-09340 from
the National Cancer Institute, NIH.
'Recipient of a Junior Faculty Research Award from the American Cancer
Society.
CANCER
RESEARCH
appear lo represent an importanl factor for the selectivity of
leucovorin rescue in vivo.
INTRODUCTION
The administration of MTX3 in conjunction with a source of
reduced folate cofactors enhances the therapeutic efficacy of
this agent in tumor-bearing animals (1, 2). The original pharma
cological premise of this rescue of host tissues from drug toxicity
was thai the added tetrahydrofolate would circumvent the MTX
block at the level of the DHFR, allowing for continued purine and
pyrimidine biosynthesis for cell division. While no obvious basis
for the demonstraled therapeutic selectivity of this approach is
apparent from these considerations alone, the success of Ihese
regimens in experimenlal systems has nonetheless led lo their
clinical implementation (3, 4).
Primarily because of its chemical stability, rescue protocols
have generally used calcium leucovorin [(6fî,S)-5-CHO-FH4].
While not a nalural cellular consliluent, the (6S)-isomer of this
compound is readily transported into cells by the MTX-lelrahydrofolate cofactor carrier (5), and, as depicted in Fig. 1, can
be assimilated into cellular folate pools following its metabolism
initially to (a) 5,10-CH+-FH4 and, subsequently, lo (o) 10-CHOFH4, Ihe one carbon donor for purine biosynlhesis, or (c) 5,10CH2-FH4, Ihe cofactor for thymidylate biosynlhesis. While (6S)5-CHO-FH4 is rapidly melabolized in tumor cells in Ihe presence
(6) and absence (7) of MTX, and can readily support cell division
(8), il is unclear whelher Ihe rates for these processes for
cofactor uplake and inlerconversion are sufficienl for cellular
replication when DHFR is inactivated by antifolates (9).
Several laboratories have reported lhal MTX and reduced
folates interacl competitively during rescue, a finding lhat cannot
easily be reconciled soley on the basis of an anabolic role for the
exogeneously supplied cofactor. While this competitive relation
ship between these folate derivatives has been attributed to
direcl binding interactions al Ihe level of Iheir shared membrane
Iransport carrier (5, 10), similar interactions have been demonslraled belween telrahydrofolale cofactors and lipophilic 2,4diaminoantifolales which bind to DHFR but penelrate cells by
mechanisms distinct from the MTX-telrahydrofolale cofactor car
rier (8, 11). This suggesls lhal olher interactions al common
cellular loci are also involved during Ihe reversal of antifolate
action by lelrahydrofolales.
3The abbreviations used are: MTX, methotrexate; 5-CHO-FH4, 5-formyl tetra
hydrofolate; 10-CHO-FH4, 10-formyl tetrahydrofolate; 5,10-CH+-FH4, 5,10-methenyl tetrahydrofolate; 5,10-CH2-FH4, 5,10-methylene tetrahydrofolate; 5-CH3-FH4,
5-methyl tetrahydrofolate; DHFR, dihydrofolate reducÃ-ase;FH2, dihydrofolate; FH4,
tetrahydrofolate; GAT, glycine (200 ^M), adenosine (100 »IM),and thymidine (10
fiM); TMQ, trimetrexate (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)-methyl]
quinazoline); HPLC, high-pressure liquid chromatography.
VOL.
46 FEBRUARY
1986
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ELEMENTS
IN LEUCOVORIN
Recent interest has focused on a potential role for competitive
interactions between MIX and folate cofactors derived from
leucovorin at the level of DHFR as a basis for the reduction of
drug activity during rescue (9,12-14). Studies from this labora
tory have shown that exogenous tetrahydrofolates in sufficient
concentrations effect a net displacement of MIX bound to DHFR
in cells (13). Significantly, for cells which have accumulated
appreciable levels of MIX polyglutamates, no net loss of en
zyme-bound antifolate occurs (13). These findings suggest a
potential role for MTX polyglutamylation as a selective cellular
determinant of leucovorin rescue in vivo. The present report
explores the molecular relationship between antifolate polyglu
tamylation, binding to DHFR, and the reversal of drug activity by
tetrahydrofolate cofactors.
MATERIALS AND METHODS
Chemicals. [3',5',7,9-3H]Folic
acid was purchased from Amersham
(Arlington Heights, IL). MTX and TMQ (glucuronate salt) were obtained
from the Drug Development Branch, National Cancer Institute, Bethesda,
MD. MTX was purified as described previously (15). (6fl,S)-5-CHO-FH4,
(6fl,S)-5-CH3-FH4, and (6fl,S)-FH4 were purchased from Sigma Chemical
Co. (St. Louis, MO). Standard folates, including 10-CHO-FH4 (16), 5,10CH+-FH4 (16), and FH2 (17) were prepared as described previously.
[3',5',7,9-3H]-(6S)-5-CHO-FH4
and unlabeled (6S)-5-CHO-FH«were pre
dialyzed fetal calf serum, 2 mw i -glutamina, penicillin
Cell numbers were determined with a Coulter counter or by direct
microscopic examination. For the growth inhibition experiments, 2.5-ml
cultures (1 x 10s cells/ml), pretreated with MTX for 3 h or continously
exposed to TMQ, as described in the text, were incubated for 48 h. After
this interval untreated cells had attained a density of approximately 1.52.0 x 106 cells/ml.
Incubation Techniques and Extraction of Cellular Folates. Cells
(approximately 4x10'
cell/ml) were incubated in specially designed
flasks under an atmosphere of 95% 02-5% C02 in complete folate-free
medium containing containing 0.02 mw 2-mercaptoethanol in the pres
ence of GAT. MTX treatment was for 3 h followed by resuspension of
NAD*
^—->N5-Methyl
FH4
Purines
FH4 «s
g for 2 min). The supematants were sampled and analyzed immediately
by HPLC as described below; the cell pellets were washed twice with
10 volumes of a 0.85% 0°Csaline solution and a portion of the washed
cells was aspirated into the tip of a Pasteur pipet and extruded onto a
polyethylene tare. After drying ovenight at 70°C, the cell pellets were
weighed directly on a Cahn model 4700 electrobalance, placed in scintil
lation vials, and digested in 0.2 ml of 1 N KOH for 1 h (70°C). After
neutralization with 0.2 ml of 1 N HCI, radioactivity was measured with a
Packard 460C liquid scintillation spectrometer using Readi Solv scintil
lation cocktail (Beckman, Irving, CA). Corrections for counting efficiencies
were made by internal standardization with tritiated toluene. In this
fashion net uptake of radiolabeled folate was expressed as nmol of
tritium per gram of dried cell pellet. Intracellular water was determined
from the difference between the wet and dry weights of the cell pellet,
less the [14C]inulin space as described elsewhere (5). A value of 0.054
±0.005 (SD) ml of intracellular water per 10e cells was determined (n =
was sealed with a serum stopper and the contents were evacuated with
a vacuum pump and flushed with nitrogen through a syringe needle. The
cellular suspension was heated in a boiling water bath for 90 s. After
cooling to 0°C, the extract was incubated overnight at 32°C under
nitrogen with a conjugase prepared from chicken pancreas (19). This
treatment resulted in an essentially complete (greater than 95%) hydrol
ysis of the polyglutamate folates to the monoglutamyl forms. A nonenzymic conversion of 10-CHO-FH4 to 5-CHO-FH4 (25%) and 5,10-O-TFH4 (less than 10%) occurred during the extraction and hydrolytic pro
cedures. While some loss of FH4 was also encountered (approximately
32%) the recoveries of 5-CH3-FH4 and FH2 were essentially quantitative
(greater than 90%). Under these conditions, 5,10-CH2-FH4 appears as
FH4. The degradation products detected both intracellularly and extracellularly following HPLC analysis (see below) were particularly prevalent
following extended incubations with the radiolabeled folate (generally 20
to 30% of total extracellular and intracellular radiolabel after 3 h). These
products were attributable primarily to oxidative degradative processes
during the incubations; the extent of this nonenzymic decomposition was
not reduced by decreasing the oxygen levels to 20%.
For analysis of total cellular folate polyglutamates, an additional portion
of washed cells was boiled in 50 ITIM sodium phosphate, pH 7.2, and
1% 2-mercaptoethanol for 3 min followed by the chemical cleavage of
the folate polyglutamates to the corresponding p-aminobenzoyl polyglu
tamates using the cleavage procedure of Fco, er a/. (20).
HPLC Analysis of Folyl Mono- and Polyglutamates. For analysis of
octadecylsilyl column (IBM Instruments, Danbury, CT). The HPLC anal
ysis consisted of a gradient of from 0 to 2.6% acetonitrile over 2 min
followed by an increase to 6.75% acetonitrile from 9 to 14 min. The flow
rate was 2 ml/min and 0.5-min fractions were collected and measured
for radioactivity as described above. The retention times for the radiola
beled cofactors were compared to those for standard folates detected
by UV monitoring at 280 nm (Table 1).
p-Aminobenzoyl polyglutamates from the reductive cleavage proce
dure were analyzed on a 1O^m Spherisorb octadecylsilyl column (Brown-
»N5,N'°-Methenyl FH4
^-» ADP, Pi
ATP
5-Formyl
and radiolabeled intracellular constituents were measured after centrifugation of samples of the cell suspension (8 x 107 cells) at 0°C(500 x
the natural folates following the enzymic hydrolysis of the polygluta
mates, the extracts were boiled for 90 s followed by centrifugation.
Samples were analyzed on an Altex Model 332 gradient liquid Chromat
ograph equipped with a Model 210 injector using a 5-^m Spherisorb
Thymidylote
Nlo-Formyl
was also for 3 h prior to
addition of the radiolabeled folate. However, TMQ exposure was contin
ued throughout the experiment. [3H]-<6S)-5-CHO-FH4 (5 /IM) was added
For the analysis of intracellular folate forms a portion of the washed
cell pellet was treated with 1 ml of nitrogen-saturated 0.1 M sodium
maléate,pH 7.2, containing 1% 2-mercaptoethanol. The centrifuge tube
(100 units/ml) and streptomycin (100 M9/ml), all purchased from Gibco
Laboratories (Grand Island, NY). Cell folate pools were depleted by
culturing the cells for several weeks in RPMI 1640 without folie acid
(Gibco Laboratories) in the continuous presence of GAT. Inocula were
made at 1 x 105 cells/ml and cell transfers were made every 2 days.
NAOH
cells in drug-free medium. TMQ treatment
4).
pared from radiolabeled folie acid and unlabeled FH2, respectively, as
described by Moran and Colman (18), except that all procedures were
conducted under a nitrogen atmosphere as described below. Other
chemicals were obtained from commercial sources.
Cell Culture. The murine L1210 leukemia was grown in a humidified
atmosphere at 37°C in RPMI 1640 medium supplemented with 10%
heat-inactivated
RESCUE
lee Laboratories, Santa Clara, CA) using a linear gradient of from 20 to
35% acetonitrile in 2.5 rriM tetrabutylammonium
phosphate in 5 mw
sodium phosphate, pH 6.5, over 15 min, followed by an isocratic elution
FH4
Fig. 1. Pathways of metabolism of (6S)-5-CHO-FH4.
CANCER
RESEARCH
VOL. 46 FEBRUARY
1986
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ELEMENTS
IN LEUCOVORIN
RESCUE
35% of control) was observed when the leucovorin concentration
was increased to 100 MM(not shown). This incomplete abolition
time
of antifolate activity by leucovorin under these conditions con
(min)2.67.58.31113.5161717.2
trasts with the essentially complete protection afforded drug-
Table 1
HPLCretention times for standard folates
Derivativep-Aminobenzoic
acid10-CHO-FH4FH,5-CHO-FH45,10-Or-FH,FH25-CH3-FH45,10-CH2-FH4Retention
treated cells by continuous exposure to GAT (96.22 ±8.68% of
control at 10 MMMTX, n = 4; also, see réf.21).
Reversal of TMQ Cytotoxicity to L1210 Cells by Leucovorin.
While the metabolism of MTX to its polyglutamyl forms inhibits
cell division in the presence of folie acid or leucovorin, a greater
reversal of the pharmacological effects of equitoxic concentra
tions of the lipophilic quinazoline, TMQ, can be achieved by
exogenous leucovorin (Fig. 3). TMQ binds avidly to DHFR (8);
however, this agent neither competes with tetrahydrofolate co
factors for cell entry (26,27), nor possesses the stuctural require
ments for polyglutamylation by the folylpolyglutamate synthetase. The pharmacological effects of TMQ are reversible follow
ing a brief drug exposure (data not shown) consistent with the
lack of metabolism of this agent to retained derivatives analogous
to MTX polyglutamates. However, continuous treatment with
this drug potently inhibits the replication of folate-depleted cells
too
incubated with 5 MMfolie acid (Fig. 3). In contrast to the findings
with MTX, at concentrations of TMQ (1-10 nM) that inhibit cell
IO"'
IO
MIX (M)
Fig. 2. Effect of MTX on growth of L1210 cells in the presence of folie acid (5
f!M)or calcium leucovorin (10 >M).L1210 cells were exposed to the indicated levels
of MTX for 3 h, resuspended into drug-free medium (at 1 x 10s cells/ml), and cell
growth was monitored after 48 h. The results are expressed as the mean ±SD
from 4 incubations in 2 separate experiments.
at 35% acetonitrile for an additional 5 min. The flow rate was 2 ml/min
and 0.5-min fractions were collected and measured for radioactivity as
described above. Radiolabeled p-aminobenzoyl polyglutamates were
Diaminoantifolates. To better understand the relationships
among the extent of reversal of antifolate activities in vitro,
extracellular drug concentration, and the effects of MTX polyglu
tamylation on leucovorin utilization, the radiolabeled (6S)-isomer
of 5-CHO-FH4 was prepared and its metabolism was evaluated
identified by comparison of their retention times with authentic standards
(Glu,.?) provided by Dr. Barry Shane (University of California at Berkeley).
The standards were detected by UV monitoring at 254 nm.
RESULTS
Effect of Leucovorin on the Cytotoxicity of MTX to L1210
Cells. Folie acid (5 UM) or leucovorin (50 nw) can meet the
anabolic requirements for tetrahydrofolates in L1210 cells pre
viously depleted of endogenous folates. Conversely, pretreat
ment of these folate-depleted cells with MTX for 3 h followed by
their resuspension into drug free medium limits cell division in
the presence of these compounds (Fig. 2). Under these condi
tions growth inhibition arises from the accumulation of MTX
polyglutamyl derivatives during the initial 3-h exposure to drug
(at 10 MMMTX, approximately 5- to 8-fold greater than the level
of DHFR). In contrast to unconjugated drug, these derivatives
are selectively retained intracellularly in the absence of extracel
lular antifolate (15, 21-25). Hence, this metabolism serves to
prolong antifolate activity in the absence of continuous drug
exposure (21, 22, 24). In Fig. 2, a 50% inhibitory concentration
of 0.59 /¿Mwas measured in the presence of 5 UM folie acid
following MTX pretreatment; this changes only slightly when 10
/¿M
of leucovorin is provided as a source of reduced cofactors
(50% inhibitory concentration, 2.2 pM). At the most inhibitory
levels of MTX (greater than 5 tiM), only a partial reversal of drug
activity was achieved by the addition of leucovorin (20-25%).
Only a slightly greater reversal of drug activity (to approximately
CANCER
RESEARCH
growth in the presence of folie acid, drug activity was completely
abolished by the addition of 10 MMleucovorin; however, at higher
TMQ levels a lesser diminution of the growth inhibition was
achieved by the reduced cofactor. At 1 MMTMQ, only 14.16 ±
3.76% (n = 4) of control levels of growth was sustained in the
presence of 10 MM leucovorin. As observed in the studies with
MTX, essentially complete protection was afforded by the pro
vision of GAT along with the antifolate (91.42 ±11.42 at 1 MM
TMQ, n = 4; not shown).
Metabolism of [3H]-(6S)-5-CHO-FH4 in the Presence of 2,4-
under conditions simulating those in the growth inhibition exper
iments. Previous reports have described extensive metabolism
of (6S)-5-CHO-FH4 in L1210 cells grown in folate replete medium
(6, 7). However, in the present studies, as described above, the
tumor cells had previously been depleted of their endogenous
folate stores prior to exposure to the radiolabeled cofactor. This
approach allows a more accurate assessment of tetrahydrofolate
TMO(M)
Fig. 3. Effect of continuous exposure to TMQ on growth of L1210 cells in the
presence of 5 UMfolie acid or calcium leucovorin (10 ^M). Cells (1 x 105/ml)were
continuouslyexposed to the indicatedlevelsof TMQ and cellgrowth was monitored
over 48 h. The results are expressedas the mean±SD from 4 separateincubations
in 2 separate experiments.
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1986
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ELEMENTS
IN LEUCOVORIN
RESCUE
metabolism without isotope dilution or metabolic perturbations
associated with endogenous folate polyglutamates.
Folate-depleted L1210 cells were incubated with 5 MM [3H](6S)-5-CHO-FH4, following which cellular folates were extracted
under nitrogen, enzymatically hydrolyzed to their monoglutamyl
forms, and analyzed by HPLC as described in "Materials and
Methods." Fig. 4 depicts the uptake and metabolism of this
IO-CHOFH4
tritiated folate in untreated L1210 cells and cells pretreated with
MTX (10 MM)for 3 h so as to form high levels of MIX polyglu
tamates, following which extracellular drug was removed. In the
untreated cells, predominantly 10-CHO-FH4 accumulated over
this interval (65% of total folates), with lower levels of 5-CHOFH4, and other tetrahydrofolates (5-CH3-FH4 and FH4). Only very
low levels of [3H]FH2 were detected intracellularly under these
nmol
conditions (less than 0.1 UM).The major effect on the intracellular
folate distributions following MTX pretreatment was the high
level of FH2 accumulated (approximately 4 MM by 30 min), con
sistent with the potent inhibition of DHFR by the MTX derivatives.
However, even in the presence of these antifolate derivatives,
substantial levels of the various tetrahydrofolates were present
as well (75% of cellular folates in the experiment shown). Similar
findings were obtained in cells continuously exposed to 1 MM
TMQ (data not shown).
After 3 h, a similar distribution of intracellular radiolabeled
cofactors was found (Fig. 5). Greater than 70% of these intra
cellular derivatives were present as polyglutamates by 3 h,
distributed among cofactor forms containing up to 4 additional
glutamates (Table 2). The total intracellular folate levels were not
significantly affected by drug treatment. Moreover, the polyglutamyl distribution was only slightly altered, with a small increase
in the tetraglutamyl form at the expense of the other derivatives,
in the presence of the antifolates.
Of particular interest was the finding after 3 h that considerable
Contro)
MTX
TMQ
Fig. 5. Total intracellular and extracellular metabolites of [3H]-<6S)-5-CHO-FH4
in L1210 cells. Folate-depleted L1210 cells (1 x 10'), either untreated or treated
with the antifolates as described in the text, were incubated with 5 MM[3H]-{6S)-5CHO-FH, for 3 h, followed by quantitation of the intracellular (•)
and extracellular
folate metabolites (Ü)as described in "Materials and Methods." Only the major
derivatives are shown since no significant FH4 or 5-CH3-FH4 was detected extracellularly.
Table 2
Effect of antifolate treatment on folate polyglutamylation
Folate-free L1210 cells treated as described in the text were incubated with [3H]
-(6S)-5-CHO-FH4 (5 MM)for 3 h, followed by determination of cellular folate polyglu
tamates as described in "Materials and Methods." The data presented are the
mean values from 3 separate experiments.
% of polyglutamate distribution3
Total folates
Treatment
1
(MM)
None
14.71
14.11
12.63
MTX (10 MM)
14.92
19.66
12.40
TMQ (1 MM)
14.74
24.91
11.51
" The numbers (1-5) designate the total glutamate
26.06
17.80
15.82
residues
29.86
17.33
36.67
13.46
37.21
10.56
associated with p-
aminobenzoate and, hence, the folate derivatives.
levels of monoglutamyl metabolites of (6S)-5-CHO-FH4 had ac
cumulated extracellularly (Fig. 5). Indeed, 89% of the total folate
metabolites derived from [3H]-(6S)-5-CHO-FH4 was present extracellularty as 10-CHO-FH4 in untreated cells following a 3-h
CONTROL
incubation. For cells pretreated with 10 MM MTX (3 h) and cells
continuously exposed to 1 MM TMQ, a small but significant
reduction in the level of 10-CHO-FH4 was observed; extracellular
10-CHO-FH4 under these conditions comprised 71 and 63%,
respectively, of the total metabolites. While considerable FH2
was detected both intracellularly and extracellularly in the pres
ence of these agents, the total level (extracellular and intracellu
lar) of this radiolabeled oxidized derivative was comparatively
small, comprising only 24 and 33%, respectively, of the total
folate metabolites in the presence of MTX and TMQ. Hence,
while the cellular metabolism of (6S)-5-CHO-FH4 is extensive
under these conditions, the utilization of this reduced cofactor
for thymidylate biosynthesis is apparently limited.
Relationship between DHFR Activity and the Reduction of
Antifolate Activity by Leucovorin. Fig. 6 presents data which
suggest that the abolition of 2,4-diaminoantifolate activity by
MTX
IO
20
30
MINUTES
Fig. 4. Uptake and metabolism of [3HJ-(6S)-5-CHO-FH4. Control cells and cells
pretreated with MTX (10 MM) for 3 h were incubated with [3H]-(6S)-5-CHO-FH4.
Total cellular tritium and cofactor distributions were determined as described in the
text. Data are presented for the accumulation of 10-CHO-FH4 (O); 5-CHO-FH, (•);
FH2 (A); and other tetrahydrofolates P). The data shown for the accumulation of
5-CHO-FH4 include the radioactivity cochromatographing with 5,10-CH*-FH4.
leucovorin is associated with the activation of cellular DHFR as
manifested by the maintenance of low levels of FH2. Here the
extent of cell growth in the presence of 10 MMleucovorin under
various conditions is correlated with the total (sum of intracellular
and extracellular) accumulation of FH2 derived from radiolabeled
CANCER RESEARCH VOL. 46 FEBRUARY 1986
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ELEMENTS
140
IN LEUCOVORIN
2.8
PERCENT GROWTH
DIHYDROFOLATE
120
2.4
i
100
2.0
l
80
1.6
ö 60
tr
u
1.2
|—
"-
40
0.8
0.4
2O
0L
10 pM
MTX
luM
TMQ
Fig. 6. Relationship between reduction of antifolate effects on cell growth by
leucovorin and DHFR activity. Cells (1x10°) were incubated with 5 pM radiolabeled
(6S(-5-CHO-FH4 for 3 h and the total level of tritiated FH2 (expressed in total nmol
accumulated as the sum of extracellular and mtracellular cofactor) was determined
as described in "Materials and Methods." The data reflect the mean ±SD from
duplicate experiments. The extent of growth inhibition was taken from the results
presented in Figures 2 and 3.
(6S)-5-CHO-FH4 over a 3-h interval. While only low concentra
tions of FH2 were formed in untreated cells (0.24 nmol), consid
erable levels of this derivative (1.46 nmol) accumulated in the
tumor cells pretreated with MTX. Similarly, at a level of TMQ (1
Õ/M)which rendered cells relatively insensitive to exogenous
leucovorin, high levels of FH2 were present (2.16 nmol). However,
under conditions in which leucovorin appreciably reversed TMQ
cytotoxicity (7.5 nw and 0.1 ¿¿M),
there was a proportional reduc
tion in the level of radiolabeled FH2 detected, suggesting a
reactivation to DHFR under these conditions.
DISCUSSION
This report explores the relationship between tetrahydrofolate
cofactor metabolism and the pharmacological activities of 2,4diaminoantifolates under conditions of leucovorin rescue in vitro.
While the original basis of the apparent selective reversal of MTX
activity by leucovorin in vivo was that an exogenous source of
reduced folates, when appropriately administered, would circum
vent the drug-induced block at DHFR, considerable experimental
evidence now supports alternative mechanisms for rescue.
The finding of competition between MTX and FH2 at the level
of the intracellular DHFR (28-30) led to the suggestion that
exogenous leucovorin might increase the intracellular FH2 pool,
resulting in a competitive displacement of a small amount of
bound antifolate sufficient to reverse the pharmacological effects
of the drug. Further studies from this laboratory demonstrated
that leucovorin, if present in sufficient concentrations, promotes
the net dissociation of underivatized MTX bound to DHFR in
tumor cells (13). While this effect could involve the formation of
cellular FH2 from the added cofactor and consequent displace
ment of drug from DHFR by this oxidized derivative, reduced
folates have also been found to displace enzyme-bound MTX in
cells even when thymidylate synthase is inhibited by treatment
with 5-fluoro-2'-dexoyuridine preventing the build-up of FH2 (13).
Further, reduced folates displace drug tightly bound to purified
DHFR (12).
Regardless of the actual folate derivative involved in the re
placement of the antifolate on the enzyme active site in cells, the
CANCER
RESEARCH
RESCUE
critical determinant of the extent of drug displacement is the
level of free antifolate in the vicintiy of DHFR at the time of
provision of the tetrahydrofolate derivative. Indeed, if this free
intracellular drug component is elevated, no significant competi
tion at the level of DHFR is observed (13). Similarly, in cells which
have accumulated MTX polyglutamates to a concentration ex
ceeding that of the DHFR, intracellular folates derived from
leucovorin do not effectively compete with the drug for enzyme
binding (13).
Consistent with these findings is the observation that the
metabolism of MTX to polyglutamyl derivatives by L1210 cells
limits the extent of rescue achieved by leucovorin in vitro. A
similar inverse relationship between the accumulation of intra
cellular MTX polyglutamates and the reversal of drug activity by
leucovorin has been suggested (31, 32). Moreover, the present
study has demonstrated that an essentially identical effect can
be obtained by the continuous exposure of cells to the structur
ally dissimilar drug, TMQ; however, at lower equitoxic levels of
TMQ, a significant diminution of antifolate activity was achieved
with leucovorin.
In this report a correlation has been presented between the
reduction of antifolate activity in tumor cells by leucovorin and
the relative activity of intracellular DHFR. For TMQ, the extent
of reduction of cytotoxicity closely paralleled the functional level
of DHFR activity, as reflected in the intracellular accumulation of
radiolabeled FH2 from exogenous (6S)-5-CHO-FH4. Hence,
DHFR reactivation, presumably arising from direct displacement
of enzyme-bound TMQ, analogous to unmetabolized MTX, is a
key event in the reversal of the pharmacological effects of this
antifolate by leucovorin. Antifolate displacement from cellular
DHFR is not possible for cells which have accumulated apprecia
ble levels of MTX polyglutamates (13). Under these conditions
cytotoxicity is sustained even when leucovorin is supplied be
cause reactivation of the enzyme is not possible. Similarly, in the
continuous presence of high levels of the TMQ, net displacement
of antifolate from DHFR is not possible, resulting in the expres
sion of cytotoxicity even when the rescue agent is provided.
On this basis, it seems that the conversion of MTX to its
polyglutamyl forms may be an important determinant of the
selectivity of leucovorin rescue in much the same fashion that
this differential metabolism appears to be a key element in the
therapeutic selectivity of MTX. These polyglutamyl derivatives of
MTX are synthesized to a much greater extent in susceptible
tumors than in bone marrow (21) and gastrointestinal cells (33,
34), contributing to a sustained intracellular level of antifolate
even as the plasma drug concentration falls. In the sensitive host
tissue, conversely, the rapid loss of underivatized MTX parallels
the plasma drug clearance (33). The elevated levels of MTX
polyglutamates that accumulate in tumor cells would also pre
clude any significant displacement of antifolate from cellular
DHFR by the provision of exogenous tetrahydrofolates. On the
other hand, the low levels of MTX polyglutamates formed in
bone marrow and gastrointestinal cells should permit a direct
displacement of bound antifolate by cellular folates derived from
leucovorin, allowing a selective restoration of endogenous tet
rahydrofolate biosynthesis in these tissues.
The finding in the present study that (6S)-5-CHO-FH4 is exten
sively metabolized to 10-CHO-FH4, even in the presence of the
antifolates, with incomplete conversion to FH2, raises the possi
bility that tetrahydrofolate utilization is somehow limited under
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1986
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ELEMENTS
IN LEUCOVORIN
these conditions. This contrasts with previous suggestions of a
rapid interconversion of cofactor forms and maximal depletion of
reduced cellular folates following drug exposure (28, 30). While
no obvious perturbations by the antifolates on either the monoglutamate or polyglutamate cofactor distributions are apparent
in these data to account for this discrepancy, this effect could
conceivably arise from unfavorable equilibria between cellular
folate derivatives (i.e., 10-CHO-FH4) and 5,10-CH2-FH4 which
limit thymidylate biosynthesis. Alternatively, these data could
reflect an inhibition of intracellular folate-dependent enzymes,
including thymidylate synthase or the purine biosynthetic en
zymes by antifolate derivatives (35, 36), or by dihydrofolate
polyglutamates which accumulate in the presence of these
agents (37). Studies are currently in progress to evaluate these
possibilités.
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46 FEBRUARY
1986
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Antifolate Polyglutamylation and Competitive Drug
Displacement at Dihydrofolate Reductase as Important
Elements in Leucovorin Rescue in L1210 Cells
Larry H. Matherly, Charles K. Barlowe and I. David Goldman
Cancer Res 1986;46:588-593.
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