[CANCER RESEARCH 37, 408-412, February 1977]
Synthesis of Methotrexate Polyglutamates in L1210 Murine
Leukemia Cells1'2
V. Michael Whitehead
Research Institute, Montreal General Hospital, Montreal, Quebec, Canada
SUMMARY
The ability of L1210 mouse leukemia cells and of a mutant
methotmexate-nesistant cell line (L1210/MTX) to synthesize
methotrexate polyglutamates was studied. Host DBA/2
mice were treated with methotmexate, after which Ieukemic
cells were harvested from ascitic fluid and levels of metho
trexate and metabolites in them were determined by Sepha
dex G-15 gel chromatography. The level of methotrexate in
Li2iO/MTX cells was 12.5 times greaten than that in L1210
cells, reflecting the increased level of dihydrofolate meduc
tase that characterizes this mutant cell line. Synthesis of
methotrexate polyglutamates in each cell line required a
dose of methotrexate (2.4 mg/kg) 10 times greater than the
dose that yielded extensive methotrexate polyglutamate
synthesis in rat liver and kidney in previous studies. Total
methotnexate polyglutamates synthesized in 4 hr with this
dose were the same in each cell line, demonstrating that
this metabolism
was not affected by differences
in the level
of dihydrofolate meductase. Methotrexate polyglutamates
comprised 47 ±20% of the total methotrexate in L1210
cells. Methotrexate diglutamate was the predominant form.
Levels of methotmexate monoglutamate and diglutamate
were similar in L1210/MTX cells, whereas methotrexate
monoglutamate was the predominant metabolite in host
liver, kidney,
and small intestine.
These differences
may
reflect differences in substrate preference of pteroylpoly
glutamate synthetase in these different tissues. Twenty-four
h r after methotmexate ad mi n istration , total methotrexate
in
Li210 cells was one-third of that at 4 hn; but the proportion
of metabolites was the same, presumably reflecting cell
death and division rather than loss of a freely exchangeable
portion of intracellular methotmexate present at the earlier
time. The affinity of methotrexate polyglutamates for dihy
drofolate reductase was found to be similar to that of meth
otnexate, providing evidence that these metabolites may be
as potent antagonists of folate metabolism as is methotrex
ate itself. Recent studies indicate that inhibition of folate
metabolism in cells requires their exposure to high levels of
methotrexate in order to achieve intracellular levels of
methotnexate greater than needed to bind to dihydnofalate
reductase. Such conditions conform to those required for
synthesis of methotrexate polyglutamates. Thus, these me
1 This
work
was
supported
by
grants
from
the
Medical
Research
Council
tabolites may play a specific molein inhibiting folate metabo
lism, distinct from the antifolate potential that they appear
to share with methotnexate.
INTRODUCTION
MTX,3 an analog of the vitamin folic acid (pteroylglutamic
acid), is an important anticancer agent. It has proved to be
particularly effective in the treatment of choniocarcinoma
(18) and of acute lymphoblastic leukemia in children (ii). It
has also proved to be of benefit in the treatment of a wide
variety of other malignant diseases. Recently, adjuvant them
apy of osteogenic sarcoma in children using exceedingly
large doses of MTX followed by citnovorum factor rescue
has been rewarded with long-term remissions (14).
MTX is a potent inhibitor of the enzyme dihydrofolate
reductase (EC 1.5.1 .3) and thereby of folate metabolism.
Classically, its cytotoxic action has been attributed to this
inhibition (3). However, recent studies have identified a mole
for a component of intracellular MTX in excess of that
portion which is tightly bound (presumably to dihydmofolate
meductase)in contributing to the inhibition of folate metabo
lism (9, 12, 21). It has been suggested that binding of MTX
to targets other than dihydnofolate neductase (9, 16) or the
attainment of mane complete inhibition of dihydrofalate
neductase by this excess intracellular MTX (12) may be
critical to the action of this agent and explain the added
benefit seen clinically with high-dose MTX treatment (14).
Synthesis of poly-'y-glutamyl metabolites of MTX has been
reported from a number of laboratories (2, 5, 10, 13, 19, 24,
25). Detailed study of this metabolism of MTX has been
carried out in mattissues (2, 24, 25). In this animal, dose
dependent synthesis of both MTX(+G1) and MTX(+G@1)
has
been observed in liven and kidney (24, 25), whereas almost
no such synthesis occurred in small intestine and thymus.
In this report, these studies have been extended to the
metabolism of MTX in L1210 murine leukemia cells, since
these cells are sensitive to MTX (21) and have served often
for the study of the activity of MTX. In addition, MTX metab
alism was studied in a MTX-resistant mutant strain, L12i0/
MTX, characterized by a high level of dihydrofolate reduc
tase (7), to see whether differences in its metabolism were
present. A preliminary report of these studies has been
presented (23).
of Canada and the Cancer Research Society.
2 Requests
for
reprints
should
be
addressed
to
Division
of
Haematobogy,
Montreal General Hospital, 1650 Cedar Avenue, Montreal, Quebec, Canada
H3G1A4.
Received July 8, 1976; accepted November 1, 1976.
408
3
The
abbreviations
used
are:
MTX,
methotrexate;
MTX(+G1),
methotrex
ate monoglutamate; MTx(+G,), methotrexate diglutamate; MTx(+G,), meth
otrexate hexaglutamate.
CANCER
RESEARCH
VOL. 37
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MTX Polyglutamates in L1210 Cells
MATERIALS AND METHODS
ng/lO9cells
36
ng/ lO9ceIIs
360
I'
Male adult DBA/2 mice bearing L1210 and L1210/MTX
A
32
320
I@
tI
leukemias in ascitic form were obtained from Dr. I. Wodin
280
I\
/‘@
sky, Arthur D. Little, Inc., Cambridge, Mass. They were me 28
I@
i
I
placed after 6 months. Leukemias were maintained by
24
240
It
I
weekly transfer to DBA/2 mice (8). For study, mice were
\
It
20
200
usually killed with ether on Day 5, and cells were harvested
from the penitoneal cavity by rinsing it with 2 to 3 ml of
16
160
sterile 0.9% NaCI solution. Cells from 2 to 4 mice were
12
120
pooled. Grossly hemorrhagic specimens were discarded.
Cells were washed 3 times with sterile 0.9% NaCI solution
8
80
and finally suspended in 4 volumes of 100 mM sodium
4
40
A
B
phosphate buffer, pH 7.0. Duplicate cell counts were made
0
0
in a Coulter Counter, Model S (Coulter Electronics,
0
8
16 24 32 40 48
8
0
16 24 32 40 48
Hialeah, Fla.) and were usually in the range of 1 to 4 x 108
tube number
cells/mb. Cells frequently clumped together during cell
Chart 1. Sephadex G-15 gel chromatograms of extracts of L1210 cells (A)
washing. This was not corrected by addition of hepanin to
and L1210/MTX cells (B), prepared 4 hr after a dose of [3H]MTX (2.4 mg/kg)
cell suspensions. Therefore, cell counts may sometimes
administeredi.p. to host DBA/2 mice. Extractswere cochromatographed
have been falsely low. Combined cytacnits and cell counts with MTX (—. ) MTX(+G,) (- - - -), and MTX(+G,) (
).
were carried out on suspensions of Li210 and L1210/MTX
cells on 9 and 16 occasions, respectively. The mean cell binder, [3H]MTX as label, and Dextran T-iO-coated charcoal
volume of Li2iO cells (553 ±162 fI) did not differ signifi
(Pharmacia) to separate bound from unbound reactants.
cantly from that of Li2iO/MTX cells (737 ±272 fI). The The ability of MTX polyglutamates to compete with [3H]MTX
mean cell volume of Li210 and L1210/MTX together was for binding to dihydrofolate neductase was measured by
671 ±254 fI. Final cell suspensions were heated in boiling
substituting them for MTX in standard curves performed by
water for 10 mm and centrifuged, and the supemnatant was direct competitive binding (15). Studies were carried out on
stored at —20°.
Preparation of extracts of host mouse tis
dihydrofolate reductase partially purified from guinea pig
sues was carried out as previously described (24).
liver (4, 15) and on dihydrofolate reductase present in by
3',5',-9-[3H]MTX (Amensham/Searle Co., Don Mills, On
sates of Li2iO and L12i0/MTX cells (1).
tania, Canada) was mixed with MTX (Ledenle Products
3H in samples was counted in an lsocap/300 Liquid Scm
Dept., Cyanamid of Canada Ltd., Montreal, Quebec, Can
tillation System (Nuclear-Chicago Corp., Des Plaines, Ill.)
ada) to obtain a specific activity of 0.1 to 0.3 @Ci/@g,
and The chromatographic behavior of unlabeled standards was
the mixture was purified by Sephadex G-i5 gel chromatog
determined spectrophotometnically on a Beckman Model
raphy (Pharmacia, Uppsala, Sweden) as previously de
DK2 recording spectrophotometem. The inhibitory biobogi
scnibed (24). [3H]MTX was usually administered to mice as a cal activity of MTX and metabalites was measured with the
single i.p. injection 4or24 hr pniorto harvesting of leukemic
folate assay organism Lactobacillus Casei (American Type
cells. MTX and metabalites in heated extracts of leukemic
Cell Culture 7469) (26).
cells and of host mouse tissues were separated by chroma
tography on columns of Sephadex G-i5 (0.9 x 55 cm), and RESULTS
were identified by cochnomatography with authentic stan
dards. MTX(+GJ, MTX(+G@), and MTX(+G13)were kindly
Synthesis of MTX Polyglutamates in L1210 and L1210/
supplied by Dr. C. M. Baugh and Dr. M. G. Nair of the MTX Cells and in Host DBA/2 Mouse Tissues. Sephadex G
Department of Biochemistry, University of South Alabama,
15 gel chromatograms of heated extracts of Li210 and
Mobile, Ala. (17). Results were expressed as ng MTX or MTX Li2iO/MTX cells revealed 2 3H-Iabeled fractions in addition
equivalent per g tissue or i0@cells. Unlike rat tissues (24, to MTX (Chart 1). These fractions were inhibitory to growth
25), chramatograms of extracts of mouse liver, kidney, and of L. casei. They corresponded in volume of elution to
small intestine contained a number of 3H-Iabeled materials
metabolites of MTX previously characterized as MTX(+G1)
in addition to but largely distinct from MTX and MTX paly
and MTX(+G@)in rat liven and kidney (24, 25). The identity of
glutamates. These materials were not present in the in
these fractions as MTX(+G@)and MTX(+G1) was confirmed
jected E3HIMTX.They were not identified. These contami
by cochnomatography with authentic standards (17) (Chart
nants accounted for as much as 20% of the 3H in some 1).
chromatograms, and they affected quantitation of MTX and
Characteristic of MTX metabolism in L12i0 cells was the
MTX polyglutamates from time to time. Fortunately, they
large proportion of MTX polyglutamates and the predomi
were neverdetected in chromatograms of L1210 and Li 210/
nance of MTX(+G@)(Chart 1A). In 6 studies of pooled Li 210
MTX cells (Chart 1).
cells, MTX polyglutamates comprised 47 ±20% of the total
The binding affinity of MTX palyglutamates fan dihydro
intracellular MTX, and the level of MTX(+G@1)
was 4 times
folate reductase was compared to that of MTX using the greaterthan that of MTX(+G1) (p < 0.05) (Table 1). The level
single-step variation of a new ligand-binding radioassay far of MTX in Li2iO/MTX cells was 12.5 times greater than that
MTX (15). This assay used dihydrofolate reductase as in L1210 cells (p < 0.001) (Chart 1B; Table 1). The level of
FEBRUARY1977
409
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V. M. Whitehead
Levels
tissuesMice
of MTX and metabolites
Table I
in L1210 and L1210/MTX
cells and in host mouse
hrlater. receiveda single i.p. injection of [3H]MTX(2.4 mg/kg) and were sacrificed 4
werealso
Cells from 2 to 4 mice were pooled and extracts were prepared.
Kidney extracts
tissue(±
pooled. Results are expressed as ng MTX or equivalent per 10@cells or g
S.D.).Levels
tissuesTissue
TotalL1210
of MTX and metabolites in cells and
MTX(+G,)
MTX(+G,)
237L1210/MTX
1554Liver
6
7
281± 162
3511 ±1483
47 ±22
166 ±97
196±145 524±
112 ±104 3789 ±
312Kidney
125Small
intestine
6
4
6
1130 ± 270
464 ± 108
1131 ± 710
370 ±96
67 ±16
35 ±13
a MTX(+G2)
No. of studies
was
absent
from
MTX
3
pooled
kidney
total MTX polyglutamates in L1210/MTX cells (278 ±197
ng/109 cells), however, did not differ significantly from the
total in L12i0 cells (243 ±161 ng/109 cells) (Table 1). Thus
both MTX-sensitive and -resistant L1210 cells appeared to
have a similar capacity to synthesize MTX polyglutamates,
although in the latter case both MTX(+G1) and MTX(+G@)
were present in roughly equal amounts. In host liver the
level of MTX(+G@)was about 6 times that of MTX(+G@)(p <
0.001). Total MTX and metabolites in mouse kidney were
about one-third of those in liver (p < 0.001). In mouse small
intestine, despite a mean level of intracellular MTX compa
rable to that in liver (Table 1), total MTX polyglutamates
constituted only 6 ±3% of the total. Again, MTX(+G1) was
the larger component (p < 0.01). Thus metabolism of MTX
appeared to be distinctive in each of the 5 tissues studied
(Table i).
Synthesis of MTX polyglutamates in L1210 and L1210/
MTX cells was dose dependent. The dose of MTX required
appeared to be 10 times greater than that which resulted in
extensive synthesis of MTX polyglutamates in rat liver and
kidney (24, 25). Thus with divided s.c. doses of [3H]MTX
totaling 0.16 and 0.24 mg/kg, almost no metabolism of MTX
was noted , whereas a divided s.c. dose of 2.4 mg/kg yielded
large quantities of both MTX(+G1) and MTX(+G@)(Table 2).
These experiments served to define an appropriate dose of
[3H]MTX for subsequent studies.
Levels of MTX and MTX palyglutamates were compared in
pooled Li 210 and Li 210/MTX cells 4 and 24 hr after a single
i.p. dose of [3H]MTX (2.4 mg/kg). This study was under
taken in part to determine whether a discernible labile intra
cellular MTX component was present 4 hr after the dose, as
concluded by Simotnak and Donsbach (20), and in part to
examine the possibility that cells surviving for 24 hr might
have metabolized MTX differently from those not surviving.
extracts
and
64 ± 48
Oa
18 ± 8
present
(22
1564 ±
537 ±
1184 ± 706
ng/g)
in
1.
Table 2
Effectsof doseof MTXon synthesisof MTXpolyglutamatesin L1210
and L1210/MTXcells
9injections
The dose of [3H]MTX was divided
and administered
as 6 or
cellsfrom at 15-mmintervals. Four hr after the 1st injection,
wereprepared
2 to 4 mice were harvested and pooled and extracts
asng
(see“Materials
and Methods―).
Resultsare expressed
MTX or MTX equivalent
per cells.Levels
i0@
of
incells
MTXand metabolites
(ng/i0 cells)
MTXCells
TotalLi210
Doseof
[3H]MTX
(mg/kg)
0.16
1010.24
2972.40
640L1210/MTX
MTX
93
277
282
7280.24
0.16
723
46472.404632
1754
MTX
(+G,)
5
‘12
68
(+G,)
3
8
290
4
8
1
7
215
366
2335
fell 78%, in kidney it fell 48%, and in small intestine it fell
92%. Differences at these times were all highly significant (p
< 0.001).
Total
levels
at 24 hr corresponded
to levels
found
previously in mattissues (24, 25), suggesting that total intra
cellular MTX was artificially and temporarily elevated at 4 hr,
particularly in liver and small intestine.
Comparison of the Affinity of MTX and MTX Polygluta
mates for Dihydrofobate Reductase. The powerful antifol
ate activity of MTX has been equated with its exceptionally
high affinity for the enzyme dihydrofolate reductase (3). To
obtain information regarding the relative potency of MTX
polyglutamates as falate antagonists, their binding affinity
far dihydnofolate neductase was compared to that of MTX.
Results obtained with dihydrofolate reductase contained in
lysates of L12i0 and Li2iO/MTX cells are shown in Chart 2.
They have been expressed as 1/fractian of [3H]MTX bound,
In 3 paired studies, the mean total 3H in pooled Li210 cells
at 24 hr was 34% of that at 4 hr (p < 0.01) (Table 3). Mean related to the quantity of unlabeled MTX or MTX polygluta
levels of MTX, MTX(+G1), and MTX(+G@)were reduced to mates present. With lysatesfrom Li210 cells, 50% reduction
39, 30, and 28%, respectively, of levels at 4 hr. Thus theme in binding of [3H]MTX was obtained with 1.i pmoles MTX
was no significant shift in proportion of MTX and metabo
and 1.2 pmabes MTX(+G@)[ratio of 2 (Chart 2.4)]. With lysate
bitesat the later time. Studies with Li2iO/MTX cells showed
from L12i0/MTX cells, 50% reduction in binding of [3H]MTX
a reduction in level of total MTX and metabolites at 24 hr to was obtained with 1.0 pmale MTX, 1.4 pmoles MTX(+GJ,
62% of that at 4 hr. Again, no major shift in proportion of 1.6 pmales MTX(+G@), and 1.0 pmole MTX(+G11). These
MTX and metabolites was discerned.
results showed that the affinities of MTX polyglutamates for
For comparison, levels of total 3H in host liven, kidney,
dihydrofalate reductase were similar if not identical to that of
and small intestine were measured 4 and 24 hr after MTX MTX. Similar results were obtained using dihydnofalate me
administration in 12 leukemia-bearing mice. Total 3H in liven ductase partially purified from guinea pig liver (not shown).
410
CANCER RESEARCHVOL. 37
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MTX Polyglutamates in L1210 Cells
3Levels
Table
of MTXand
i.p.injection
metabolitesin L1210and L1210/MTXcells 4 and 24 hr after a single
miceResults
of (3HJMTX (2.4 mg/kg)
are expressed
as ng MTXcells.Levels
or equivalent
into host DBA/2
per i0@
of MTX and metabolites in cells (ng/i09 cells)
No. of
TotalLi2iO
Cells
Time
studies
(hr)
8i3
3
4
24
387 ±143
149 ± 28
2
8442
4
24
3602 ±765
2199± 4
•
66Li2iO/MTX
10
10
8
8
MTX
6
4
0
0.
C)
2
0
0
0
2
4
6
8
0
10
2
4
6
8
10
pmoles
Chart2. Comparison
of theaffinityof MTXandMTXpobyglutamates
for
dihydrofobate reductase present in lysates of (A) L1210 cells and (B) L1210/
MTX cells, using a radiochemical bigand-binding assay for MTX (1, 15).
Incubation mixtures consisted of 0.9 ml of 0.01 M potassium phosphate, 0.15
M KCI buffer (pH 6.0) containing 1 mg albumin and 0.1
@moIeNADPH per ml,
0.1 ml MTX or MTX polyglutamate solution containing 2.2 to 90.0 pmoles/ml,
and 0.1 ml of [3H]MTX containing 15 pmoles/mb. Incubation was started with
the addition of 0.1 ml binder (lysate) preparation (binding capacity, 1 to 1.2
pmoles MTx). After 10 mm of incubation at 4°,the reaction was stopped by
theadditionof 1.0mlof asuspension
of 2.5%charcoaland0.5%DextranT
10. Standard curves prepared with MTX (—, •),with MTX(+G,) (- - - -),
with MTX(+G,) (—. _.), and with MTx(+G6) (
, 0) are shown.
These studies were carried out at pH 6.0. Essentially identi
cab results were obtained when the affinities of MTX and
MTX(+G@)far dihydrofolate reductase from Li 210/MTX and
guinea pig liver were compared at pH 7.0 and 8.0.
DISCUSSION
MTX polyglutamates were previously identified in rat tis
sues (2, 24, 25). In these studies, MTX(+GJ and MTX(+G1)
were recognized as 3H-labeled materials that produced fol
ate-reversible inhibition of growth of the fobate assay orga
nisms L. casei and Streptococcus fecalis. They were identi
fied as pobyglutamates by the demonstration that they chro
matognaphed as MTX after incubation with a source of
pteroylpalygbutamate hydmobase(‘y-glutamyb
carboxypepti
dase, falate conjugase) (2, 24, 25). Their identities were
established by cochromatography using authentic stan
dards of MTX(+G@)and of MTX(+G11)(17). The demonstra
tion of synthesis of MTX(+G1) and of MTX(+G@) in both
L12i0 and Li2iO/MTX cebls(Chart i)extendsthe number of
tissues in which such metabolism of MTX has been found to
occur (2, 5, 10, 13, 19, 24, 25). Not surprisingly, synthesis of
MTX polyglutamates was also found in host mouse tissues
(Table 1), confirming previous observations (22).
The present results were not entirely anticipated. Be
cause rat small intestine did not synthesize MTX pabygluta
mates (24, 25) (and small intestine is exquisitely sensitive to
MTX toxicity), it appeared likely that L12i0 cells, themselves
MTX(+G,)
66 ± 6
20 ± 8
MTX(+G2)
275 ±169
77 ± 31
727 ±
246 ±
277 ±29 248 ± 50 4127 ±
154±36 211± 52 2563± 84
sensitive to MTX toxicity, might backthis metabolic pathway
as well. Indeed, almost no metabolism of MTX occurred in
either Li 2i 0 or Li 210/MTX cells with doses previously used
in the rat (Table 2). However, this may have been due in part
to failure of MTX to accumulate in sufficient amount in
ascitic fluid after s.c. injection (20). When a dose of MTX
(2.4 mg/kg) was used that was comparable to the dose
recommended for the reduction of the L12i0 leukemia cell
mass (3.0 mg/kg host weight) (8), extensive metabolism of
MTX was indeed seen, both with divided s.c. administration
and with single i.p. doses. Synthesis of MTX pobyglutamates
also occurred in mouse small intestine with these doses.
Thus, sensitivity to MTX toxicity did not equate with failure
to synthesize MTX palyglutamates. Nor could resistance to
MTX toxicity, which characterizes the L1210/MTX mutant
cell line, be attributed to differences in MTX pobyglutamate
synthesis, since the total quantity of MTX polyglutamates
was the same in both cell lines.
MTX(+G@1)
was the predominant metabolite in Li2iO cells.
However, MTX polyglutamates larger than MTX(+G1), e.g.,
MTX(+G3), did not separate well from it under the condi
tions of chromatography used in the present study. These
require a large column to be visualized (unpublished abser
vation). Therefore, it is possible that L12i0 cells synthesized
some MTX polyglutamates containing more than 2 addi
tionab -y-glutamyb residues (Chart iA). In contrast, the major
metabolite of MTX in other tissues studied to date has been
MTX(+G1). Each of the tissues studied here showed a dis
tinctive pattern of distribution of MTX and metabolites. It
may be speculated that the predominance of MTX(+G@)in
Li210 cells reflects a difference in the enzyme pteroybpoly
glutamate synthetase in this cell with regard to substrate
preference. Further, it is possible that differences in metab
obism of MTX in different tissues may reflect differences in
pteroylpolyglutamate synthesis in these tissues as well. Lev
ebs of total MTX pobyglutamates in Li2iO and Li2iO/MTX
cells were the same despite the high level of MTX in the
latter, reflecting no doubt the high level of dihydrofolate
reductase that characterizes this mutant (7). It was con
cbuded that synthesis of MTX polyglutamates was not no
ticeably affected by this bargedifference in level of dihydro
fobate reductase.
The concept of “free―
intracellular MTX, or of bevels of
MTX in cells in excess of that required to bind to dihydrofol
ate reductase (9, 12, 21), has taken on importance as a
result of new therapeutic initiatives, with the use of high
dose MTX (14), and following a number of studies which
have credited this intracellular portion of MTX with a critical
role in the inhibition of folate metabolism (9, 12, 16, 21).
FEBRUARY1977
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411
V. M. Whitehead
ate in Tissues and Biological Fluids. Cancer Res., 35: 2033-2038, 1975.
Sirotnak and Donsbach (20) reported levels of MTX in Li 210
2. Baugh, C. M., Krumdieck, C. L., and Nair, M. G. Polygammaglutamyl
cells 4 hr after an i.p. dose of MTX (3.0 mg/kg) to be greater
Metabolites of Methotrexate. Biochem. Biophys. Res. Commun. , 52: 27than the bevel of dihydrofolate reductase. This bevel had
34, 1973.
3. Bertino, J. R. The Mechanism of Action of the Folate Antagonists in Man.
fallen by 24 hr, and the fall was attributed to the lass of
Cancer Res., 23: 1286-1306, 1963.
freely exchangeable MTX present within these cells at the
4. Bertino, J. R., Iannotti, A. T., Perkins, J. P., and Johns, 0. G. Dihydrofol
ate Reductase from Guinea Pig Liver and Small Intestine. Biochem.
earlier time. If this were so, one would have expected a
Pharmacol., 15: 563-571 , 1966.
disproportionate fall in level of MTX compared to MTX
5. Brown, J. P., Davidson, G. E., Weir, 0. G., and Scott, J. M. Specificity of
polyglutamates in Li210 cells at 24 hr in this study. In fact,
Fobate-y-L-glutamate Ligase in Rat Liver and Kidney. Biosynthesis of
Poby-y-L-glutamates of Unreduced Methotrexate and the Effect of Metho
the proportion of MTX and metabolites was the same at
on Fobate Polyglutamate Synthesis. Intern. J. Biochem., 5: 727both times. These results do not define a so-cabled “labile― trexate
733,1974.
portion of MTX at 4 hr, nor do they explain the subsequent
6. Coward, J. K., Parameswaran, K. N., Cashmore, A. R., and Bertino, J. R.
7,8-Dihydropteroyl Oligo-'y-L-glutamates: Synthesis and Kinetic Studies
fall. It is possible but unlikely that MTX and metabolites
with Purified Dihydrofobate Reductase from Mammalian Sources. Bio
were released from living Li2iO cells at the same rate.
chemistry, 13: 3899-3903, 1974.
7. Friedkin, M., Crawford, E., Humphreys, S. A., and Gobdin, A. The Associ
However, this was not seen with rat liver (24, 25). It is more
ation of IncreasedDihydrofolateReductasewith AmethopterinResist
likely that the fall in bevelof MTX and metabolites reflected
ance in Mouse Leukemia. Cancer Res., 22: 600-606, 1962.
the combined effect of cell death and of division and growth
8. Geran, R. I., Greenberg, N. H., Macdonald, M. M., Schumacher, A. M.,
and Abbott, B. J. Protocols for Screening Chemical Agents and Natural
of the surviving population.
Products against Animal Tumors and Other Biological Systems, Ed. 3.
Jacobseta!. (13) studied the ability of MTX(+GJ to inhibit
Cancer Chemotherapy Rept., 3(Part 3): 29—40,
1972.
the activity
of dihydrofobate
reductase
purified
from
L12i0
cells. They found it to be as potent as MTX. In fact, it
appeared to be a more potent inhibitor than MTX when
studied
at pH
8.5.
Further,
they
showed
that
growth
of
Li2iO cells in culture was inhibited equably by MTX and
MTX(+G1) and that MTX(+G3) had antitumor activity simi
bar to that of MTX in viva in prolonging the life-span of
BALB/c x DBA/2 F1mice bearing the Li2iO leukemia. The
demonstration in this study that MTX pobyglutamates bound
as tightly to dihydmofolate reductase as did MTX (Chart 2)
provides further evidence that these metabobites are as pa
tent antifolates as is MTX and that decreased membrane
transport of MTX polyglutamates appears to be the major
reason for their lesser toxicity in whole-cell systems such as
S. feca!is (17). These results also demonstrate the insensi
tivity of the MTX binding site on dihydrofolate neductase
regarding the number of glutamyb residues present. A simi
bar observation
has been
made
regarding
this
enzyme
and
poly-y-glutamyb derivatives of dihydrofolate (6). Lastly, this
study showed that measurement by ligand-binding madioas
say (15) of total tissue MTX and metabobites can be carried
out with a single standard curve and without the need to
isolate constituents.
It has been speculated that binding of MTX to intracellular
sites of bower binding affinity than that for dihydrofobate
reductaseisrequired
forinhibition
offobate
metabolism(9).
Suggested targets have been thymidybate synthetase (EC
2.1 .1.b), and the fobate-dependent enzymes involved in pu
minesynthesis. Present results would suggest that MTX po
lyglutamates comprise part of this component of intracelbu
lamMTX.
9. Goldman, I. D. Analysis of the Cytotoxic Determinants for Methothrexate
(NSC-740): A Role for “Free―
Intracellular Drug. Cancer Chemotherapy
Rept., 6: 51—61,
1975.
10. Hoffbrand, A. V., Tripp, E., and Lavoie, A. Synthesis of Folate Polygluta
mates in Human Cells. Clin. Sci. Mol. Med., 50: 61-68, 1976.
11. Holland, J. F., and Glidewell, 0. Oncologists Reply: Survival Expectancy
in Acute Lymphocytic Leukemia. New EngI. J. Med., 287: 769-777, 1972.
12. Jackson, R. C., Hart, L. I., and Harrap, K. R. Intrinsic Resistance to
Methothrexate of Cultured Mammalian Cells in Relation to the Inhibition
Kinetics of Their Dihydrofolate Reductase. Cancer Res., 36: 1991-1997,
1976.
13. Jacobs, S. A., Adamson, A. H., Chabner, B. A., Derr, C. J., and Johns, 0.
G.Stoichiometric
Inhibition
ofMammalian
Dihydrofolate
Reductase
by
14.
15.
16.
17.
18.
SequentialUseof Methotrexateand Actinomycin0 in the Treatmentof
19.
20.
21.
22.
23.
ACKNOWLEDGMENTS
The author thanks Marlene Dupont, Maureen McCormack, and Viviane
Nicolet for technical assistance; Charles Baugh and M. G. Nair for the kind
provision of MTX polyglutamates;and Bart Kamenfor his advicewith the
radioassay.
24.
25.
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CANCER RESEARCH VOL. 37
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
Synthesis of Methotrexate Polyglutamates in L1210 Murine
Leukemia Cells
V. Michael Whitehead
Cancer Res 1977;37:408-412.
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