Download Role of Folylpolyglutamate Synthetase in the

[CANCER RESEARCH 48, 2426-2431. May I, 1988]
Role of Folylpolyglutamate Synthetase in the Regulation of Methotrexate
Polyglutamate Formation in H35 Hepatoma Cells1
Thomas B. Johnson, M. G. Nair, and John Galivan2
Wadswonh Center for Laboratories and Research, New York State Department of Health, Albany, New York [T. B. J., J. G.]; and Department of Biochemistry, University
of South Alabama, Mobile, Alabama /M. G. N.J
ABSTRACT
The effect of culture conditions on the glutamylation of methotrexate
by intact H35 hepatoma cells and folylpolyglutantate synthetase (FPGS)
activity in the corresponding crude extracts has been examined. The rate
of cellular glutamylation of methotrexate observed in rapidly dividing
cultures was 4-fold higher than confluent cultures, and was accompanied
by an increase in extract FPGS activity (2.2-fold). The depletion of
cellular folates produced comparable increases in both cellular metho
trexate glutamylation and extract FPGS activity (approximately 1.8fold). Near-quantitative reductions in cellular methotrexate glutamylation
were caused by media additions of reduced folates and methotrexate to
confluent cultures of wild-type and folate-depleted H35 cells. However,
these produced relatively modest reductions in FPGS activity in the
correspondingcrudeextracts (approximately 50%). Methionine exclusion
resulted in a greater than 50% decrease in FPGS activity in crude extracts
of these cells compared to extracts of control cultures. The combination
of methionine exclusion and folinic acid addition lowered the FPGS
activity to less than 25% that of control. The data suggest that the
changes in the glutamylation rate of methotrexate in whole cells due to
culture conditions such as folate restriction, reduced folate addition,
methionine exclusion, and growth state are at least in part a consequence
of alterations in FPGS activity. This conclusion is consistent with the
proposition that the metabolism of slow-acting substrates for FPGS
(such as 4-amino antifolates and their corresponding polyglutamates)
may be sensitive to changes in enzyme levels or activity (Cook et al.,
Biochemistry, 26: 530-539, 1987). Analysis of the products formed by
FPGS from extracts using methotrexate as the substrate revealed no
significant amounts of polyglutamate species higher than 4-MI.-1 (M 'II,PteGluj. In contrast, when using the thymidylate synthase inhibitor V"propargyl-5,8-dideazafolic acid as the starting substrate under identical
assay conditions, FPGS from extracts catalyzed the formationof predom
inantly long chain polyglutamate derivatives (Gliu and higher). These
results reflect the relative efficacy of methotrexate and W'-propargyl5,8-dideazafolic acid, as well as their polyglutamate derivatives, as sub
strates for FPGS.
INTRODUCTION
FPGS3 (EC 6.3.2.17) catalyzes the conversion of folates and
folate analogues into 7-polyglutamate derivatives. The polyglutamylation of folates (1-5) serves several physiological func
tions. Folylpolyglutamates appear to be the preferred substrates
for many of the folate-requiring enzymes of one-carbon metab
olism (1,6), and have been shown to be more efficiently "chan
neled" than monoglutamates from one active site to another in
folate-requiring multienzyme complexes (7). Polyglutamate de
rivatives of folates and methotrexate are more avidly retained
in cells than their monoglutamate counterparts, and thus the
Received 11/3/87; revised 2/1/88; accepted 2/8/88.
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.
1This work was supported by NIH Grants CA25933, CA32687, and CA34314
by the National Cancer Institute, USPHS/Department of Health and Human
Services.
1 To whom requests for reprints should be addressed.
3 The abbreviations and trivial names used are: FPGS, folylpolyglutamate
synthetase; 4-NH2-10-CH3-PteGlu, methotrexate (MTX) or 4-amino- 10-methylpteroyl glutamic acid; PDDF, /V'°-propargyl-5,8-dideazafolic acid, N-\4¡(2-amino-4-hydroxy-6-quinazolinyl)methyl]prop-2-ynyl amino|benzoyl-L-glutamic acid, also known as CB3717.
FPGS-catalyzed synthesis of methotrexate polyglutamates is a
major determinant in the cytotoxicity of this antifolate (1,814). Methotrexate polyglutamates have been shown to be at
least as effective as methotrexate against the target enzyme,
dihydrofolate reducÃ-ase (8, 9, 11, 12, 15, 16), and therefore
their prolonged cellular retention affords them greater cytotoxic
potential than the parent drug.
Mammalian FPGS has been partially purified from rat (17),
mouse (18), and beef liver (19), and has only recently been
purified to homogeneity from hog liver (20). While studies
utilizing these preparations have been extremely useful in elu
cidating the chemical and physical properties of the enzyme, a
limited amount of data are available as to its mode of activity
in cells and its role in regulating the balance of cellular polyglu
tamates of methotrexate. Furthermore, relatively few investi
gations on FPGS have used tumor cells as an enzyme source.
Finally, there appear to be marked differences in the ability of
FPGS to glutamylate methotrexate in whole cells versus exper
iments with isolated enzymes, especially with regard to the
distribution of polyglutamate products (13, 14, 21-25).
Previous studies in our laboratory (26, 27) have shown that
the ability of H35 hepatoma cells to glutamylate methotrexate
can be altered by changes in growth state and culture conditions.
In an effort to determine if these alterations are a consequence
of changes in FPGS activity, as well as to gain additional insight
into the process of glutamylation in intact cells, we have quantitated the FPGS activity in extracts of H35 hepatoma cells
grown under those conditions which caused the greatest differ
ences in cellular glutamylation of methotrexate. We have also
attempted to understand what factors account for the differ
ences in polyglutamate product distribution with isolated en
zyme compared with that seen in cultured tumor cells by
evaluating the glutamylation of methotrexate and the thymi
dylate synthase inhibitor PDDF (28-30). It is envisioned that
a better understanding of the role of FPGS in the glutamylation
of these antifolates will contribute to an enhanced understand
ing of their activities as chemotherapeutic agents.
MATERIALS AND METHODS
Materials. Swims' medium S-77, folie acid-free Swims' medium,
methionine-free Swims' medium, fetal calf serum (dialyzed and undialyzed), and horse serum (dialyzed and undialyzed) were purchased
from Grand Island Biological Company. L-[2,3-3H]Glutamic acid
(NET-395, 25 Ci/mmol) was obtained from New England Nuclear
(Boston, MA). Cellulose power (CF 11) and DEAE-cellulose (DE 52)
were purchased from Whatman. Methotrexate, [3',5',7-3H]methotrexate, and 4-NH2-10-CH3-Pte-[G-3H]Glu2 were purchased from Moravek
Biochemicals (La Brea, CA). Methotrexate polyglutamate standards
were the kind gift of the National Cancer Institute. PDDF, PDDF
polyglutamates, and [14C]PDDF labeled with [G-MC]glutamate were
synthesized by the method of Nair et al. (31). Thymidine, hypoxanthine,
and ATP were purchased from Sigma. Folinic acid was supplied by
ICN Pharmaceuticals, and 5-methyltetrahydrofolic acid was prepared
as previously described (32). Methotrexate and all folates were purified
by DEAE-cellulose chromatography prior to use (32). All other chem
icals were reagent grade.
2426
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REGULATION BY FOLYLPOLYGLUTAMATE SYNTH ETASE
Cell Culture. H-11-E-C3 cells derived from the Reuber H35 rat
hepatoma (referred to as H35 cells) were grown on 60- or 100-mm
Falcon dishes in a 5% CO.- atmosphere and were subcultured weekly.
The culture medium was Swims' medium S-77 supplemented with 5%
fetal calf serum, 20% horse serum, and 4 HIMglutamine, which was
routinely changed at 72 and 120 h after plating. The cells were released
from the dishes with 0.05% trypsin, counted with a Model ZBI Coulter
counter and diluted to a seeding density of 5 x 10' cells/ml. Folatedepleted H3S cells were generated as before (26) and cultured in a
similar fashion, but in folate-free Swims' medium S-77 supplemented
with 50 /¡Mthymidine, 50 /IM hypoxanthine, 5% dialyzed fetal calf
serum, 20% dialyzed horse serum, and 4 m\i glutamine.
Preparation of Crude Extracts. Wild-type or folate-deplete H35 cells
were seeded in 100-mm Falcon dishes at a density of 6 x 10s cells/
dish, and unless otherwise noted, were grown for 96 h prior to changing
to the desired culture medium for experiments, which was accomplished
by washing each 100-mm plate with 5 ml of the desired medium lacking
serum and replenishing with 5 ml of the same medium. After the
indicated time, the cultures were cooled on ice, the medium was
removed, and each dish was washed with 3- x 4-ml ice-cold phosphate
buffered saline (pii 7.6). The cells were scraped into ice-cold 0.5 M
Tris Cl (pH 8.85), 0.2 M 2-mercaptoethanol (approximately 1 ml
buffer/15 mg total cell protein) and lysed by freeze-thawing (3x) in dryice/ethanol. The debris was removed by centrifugation (4"C) at 27,000
x g for 30 min. Following removal of an aliquot for protein determi
nation by the Lowry method (33), the supernatant was stored at —
70"C.
The FPGS activity of the crude extracts stored in this manner did not
change for 6 weeks. The activity of a particular extract was typically
determined on the day following preparation.
Ammonium Sulfate Fractionation. Solid ammonium sulfate was added
to the crude supernatant over 20 min at 0°Cwith stirring until 35%
saturation was reached. The solution was stirred at the same tempera
ture for l h and centrifuged (4"( ') for l h at 27,000 x g. The supernatant
was discarded, and the pellet was suspended in a minimum volume of
20 HIMpotassium phosphate buffer (pH 7.5), 50 HIM2-mercaptoethanol, 20% glycerol. Following protein determination (33) the sample
was divided into 1.5-ml portions and stored at —70°C.
FPGS activity
was determined without dialysis.
Enzyme Assays. Folylpolyglutamate synthetase was assayed accord
ing to the method of McGuire et al. (17). Methotrexate was used
routinely at saturation (250 /IM) as the substrate for FPGS activity
determinations (21). The stock solution of glutamate (50 in M) was
prepared by combining 50 mM sodium glutamate (pH 7.5), 150 HIM
sodium glutamate (pH 9.0), and L-[2,3-3HJglutamate (NET-395) from
New England Nuclear in a 3:1:2 volume ratio. The specific radioactivity
of the glutamate stock solution was 12-14 x 10" dpm/Vmol. Each
assay solution contained, in a final volume of 0.250 ml at pH 8.4, 100
HIMTris-Cl (pH 8.85), 10 mM ATP, 20 mM MgO2, 20 mM KC1, 100
HIM 2-mercaptoethanol, 4 mM [3H]glutamate, and methotrexate or
PDDF. Following termination of the reaction with addition of l-ml
ice-cold 5 HIMNa-glutamate (pH 7.5), 25 mM 2-mercaptoethanol, the
unreacted [3H]glutamate was separated from the polyglutamates by
DEAE-cellulose column chromatography (column buffer 10 mM TrisCl (pH 7.5), 110 mM NaCl, 25 mM 2-mercaptoethanol). The DEAE
minicolumns were then washed with 2 ml water prior to elution of the
radioactive polyglutamates with 3 ml 0.1 N HC1. The FPGS activity
for a particular extract was determined by assaying the extracts in
duplicate for l h at 400 ;<gof added protein and for 2 h at 200 ¿tgof
added protein. One-third of the acid eluant was used for counting and
the remainder was lyophilized for product analysis. -y-GIutamyl hydrolase (conjugase) activity was monitored according to the method of
Samuels et al. (34).
Methotrexate and PDDF Glutamylation in Intact H35 Hepatoma
Cells. Wild-type H35 hepatoma cells were seeded on 60-mm Falcon
dishes at a density of 2 x IO5cells/dish and grown in culture for 96 h.
The medium was changed to folate-free Swims' medium S-77 with
with 4- x 4-ml ice-cold isotonic saline containing 10 mM potassium
phosphate, pH 7.4. Four dishes were used for each experimental point.
Two were washed separately with l N NaOH to determine the total
intracellular polyglutamates and cellular protein. The two remaining
dishes were pooled in 2 ml distilled water, heated in a boiling water
bath for 10 min, and lyophilized for product analysis.
Product Analysis. Methotrexate polyglutamate product distribution
was determined by high-pressure liquid chromatography as previously
described (27). The same system was employed for the analysis of the
polyglutamates of PDDF except that a linear gradient of 4 to 24%
acetonitrile in 0.1 M sodium acetate over 40 min was used.
Charcoal Controls. A crude extract of folate-deplete H35 hepatoma
cells which had been exposed to 20 //\i folinic acid for 24 h was divided
into two 0.5-ml portions and placed on ice. Sufficient ice-cold 2.5%
dextran-treated charcoal (35) to bind 10 nmol of folate was added to
one extract, while an equal volume of ice-cold water was added to the
control. After gentle mixing both extracts were centrifuged (4°C)for
10 min at 12,000 rpm (Beckman Microfuge B), and the supernatants
were each assayed for FPGS activity.
RESULTS
Conditions for Determination of FPGS Activity. In order to
determine FPGS activity in extracts of H35 cells, duplicate
incubations of 400-/xg extract protein for l h and 200 /¿g
protein
for 2 h were conducted. Under several conditions shown in
Table 1, this combination of assays exhibited equivalent product
formation with 250 J¿M
methotrexate as substrate. These results
are consistent with linearity with respect to time and protein
concentration. However, extension of assays to longer time
periods with either amount of protein caused a loss of linearity.
Shorter incubation times could be utilized, but these could
compromise the sensitivity of the assay, especially with less
active extracts. Thus, we routinely used the assay format de
scribed in Table 1 for measuring the activity of FPGS in cell
extracts.
The linearity of the assay suggested that 7-glutamyl hydrolase
(conjugase) was not interfering with the measurement of meth
otrexate polyglutamate formation in assays of crude extracts.
To determine the relative activity of 7-glutamyl hydrolase under
the conditions of the FPGS assay, the hydrolysis of 200 pmol
of 4-NH2-10-CH3-Pte-[G-3H]Glu2 (a typical amount formed in
an assay) was measured by incubation with 400 ¿tgof extract
for l h or 200 n%of extract for 2 h at pH 8.4. High-performance
liquid chromatography analysis (34) showed that in each case
less than 0.3% of the substrate had been converted to the
monoglutamate. Thus 7-glutamyl hydrolase, which has a rela
tively low activity in these cells (36) did not interfere with the
measurement of FPGS under these conditions.
Methotrexate was compared to tetrahydrofolate as a sub
strate for FPGS from crude extracts under the standard assay
Table I Product formation from ['HJglutamate and methotrexate with FPGS
from extracts of wild-type H35 hepatoma cells with respect to time and protein
concentration
The results are the average of identical duplicate experiments. Confluent and
log phase cells were harvested after 96 and 48 h in culture medium, respectively.
Extracts of cells lacking serum in culture were harvested after exposure of cells
grown as described in "Materials and Methods" to Swims' S-77 medium + insulin
(10 mU/ml) * 4 mM glutamine for 24 h (total culture time, 120 h).
in
Culture
conditionConfluentConfluent—Serum—SerumLog
time(min)601206012060120Protein
added
(^g)400200400200400200[3H]Glutamate
corporated
(pmol)260251285320520621
insulin (10 mU/ml), and after 24 h the cells were incubated for the
indicated time period with 10 /IM [3H]methotrexate or 10 /¿M(14C]PDDF at a specific radioactivity of 4 x IO4 dpm/nmol. Polyglutamate
formation was terminated by cooling the cultures on ice and washing
2427
phaseLog
phaseIncubation
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REGULATION BY FOLYLPOLYGLUTAMATE SYNTHETASE
conditions. At saturating concentrations (250 and 35 /IM for
methotrexate and tetrahydrofolate, respectively) methotrexate
typically incorporated approximately 1.8-fold as much gluta
mate as did tetrahydrofolate (data not shown).
Alterations in Cellular MIX Glutamylation and Extract FPGS
Activity as a Function of Growth State and Culture Condition. A
comparison of the rate of glutamylation of methotrexate by
intact H35 hepatoma cells and FPGS activity in crude extracts
of the same cultures is shown in Table. 2. The greater rate of
cellular glutamylation of methotrexate observed in rapidly di
viding cultures (>4-fold higher than confluent cultures) was
accompanied by an increase in extract FPGS activity (2.2-fold).
The depletion of cellular folates produced comparable increases
in both cellular methotrexate glutamylation and extract FPGS
activity (approximately 1.8-fold). The addition of exogenous
reduced folates or methotrexate to confluent cultures of wildtype and folate-depleted H35 cells, which in some cases caused
near-quantitative reductions in cellular methotrexate glutamy
lation, produced relatively modest reductions in FPGS activity
in the corresponding crude extracts (approximately 50%). To
determine if the reduction in FPGS activity in the cytosol of
the folate-treated cells represented substrate competition be
tween the added folate in the cell extracts and the substrate,
methotrexate, an extract of folate-deplete cells which had been
exposed to 20 /¿M
folinic acid for 24 h was treated with 2.5%
dextran-treated charcoal and reassayed for FPGS activity. No
significant change compared to a charcoal-untreated extract
was observed, indicating that an increase in cellular folates
following exposure to folinic acid was not altering the apparent
FPGS activity in extracts.
The relationship between extract FPGS activity and growth
state of wild-type H35 cells was further investigated by meas
uring the enzyme activity in extracts of cells harvested at various
points in the normal growth cycle. The results of this study are
depicted graphically in Fig. 1. Extract FPGS activity was shown
to gradually increase for the first 48 h and then peak sometime
between 48 and 54 h before returning to the initial level in
extracts of confluent cells (500-600 pmol/h/mg).
Alterations in FPGS Activity in Extracts of Folate-Deplete
1135 Hepatoma Cells Deprived of Methionine. Earlier studies
1500
1000
co=)40
Î_jUJü£
co
O
1I•-8060cctu
0-
500
-r1•
20
._'.j—Õ1'"X,,\1
24
48
54
72
144
CULTURE (hrs)
Fig. 1. The relationship between extract FPGS activity (H) and growth state
(O) of wild-type H35 cells. The cells were seeded at a density of 5 x IO4cells/ml
(2 X 10s cells/60-mm dish), cultured for the indicated time in Swims' S-77
medium, 5% fetal calf serum, 20% horse serum, and 4 mM glutamine. Cell
numbers were determined by visual counting using a Zeiss model E invertoscope
equipped with a reticle. Preparation of extracts of these cultures and measurement
of FPGS activity were accomplished as described in "Materials and Methods."
The results are the average of three experiments except for the 24-h assay for
FPGS which is the mean of two observations.
Table 3 Effect ofmethionine exclusion and folate addition on FPGS activity in
extracts of folate-deplete H35 hepatoma cells
Data are presented as the mean of at least four separate measurements under
linear assay conditions with standard deviation. Folate-depleted cells were grown
in culture for 96 h. Media was removed and the cultures were rinsed with 4 ml
sterile Hanks' solution, and then exposed to folate-free Swims' S-77 medium +
glutamine (4 mM), insulin (10 mU/ml), ±methion ine (100 /IM), + folinic acid
(20 MM),or 5-methyltetrahydrofolate (20 /¿M)
for 18 h. Rinsing and exposure was
then repeated and the cells were harvested 24 h later.
FPGS activity
(pmol/h/mg)
Control
-Methionine
—Methionine + folinic acid
-Methionine + 5-methyltetrahydrofolate
1103 ±93
476 ±96
245 ±60
515°
" Two determinations.
Table 2 Alterations in the glutamylation of methotrexate by intact H35
hepatoma cells and FPGS from crude extracts by growth state and culture
conditions
Log phase cells were harvested after 48 h in culture medium. Cells at confluence
were exposed to folinic acid (20 /IM), 5-methyltetrahydrofolate (20 pM), or
methotrexate (20 MMin whole cell experiments, 10 pM for cells to be made into
extracts) for 24 h prior to incubation with [3H]methotrexate or harvesting. In
whole cell experiments, the indicated additions were removed 30 min before the
4-h incubation with labeled methotrexate. For all the conditions tested, intact
H35 cells synthesize methotrexate polyglutamates linearly with respect to time
for 4 h at a media concentration of 10 MM(26). The results for the activity of
FPGS in extracts are the average of two separate duplicate assays performed as
described under "Materials and Methods." The average standard deviation when
comparing extracts of cells grown under identical conditions is 15% (n = 55).
Wild-type
cellsLog H35
phaseControl
(confluent)+
acid+
Folinic
MethotrexateFolate-deplete
cellsControl H35
(confluent)+
acid+
Folinic
5-Methyltetrahydrofolate-tMethotrexateWhole
* Data from Ref. 26.
from this laboratory have shown that the exclusion of methionine from the culture media caused nearly a 70% reduction in
the glutamylation of methotrexate by intact folate-deplete H35
hepatoma cells (14). As can be seen in Table 3, methionine
exclusion resulted in a greater than 50% decrease in FPGS
activity in crude extracts of these cells compared to extracts of
control cultures. The combination of methionine exclusion and
folinic acid addition lowered the FPGS activity to less than
25% that of control. The combination ofmethionine exclusion
and 5-methyltetrahydrofolate addition reduced the activity to
approximately half that seen with extracts of control cultures.
cellMTX
Analysis of Products Formed from Methotrexate and PDDF
gluta
FPGSactivity(pmol/h/mg)14266383463621198775616675
mylation*(pmol/4
in Intact r135Hepatoma Cells and by FPGS from Crude Extracts
h/mg)211470.212837215Extract
or Resuspended 0-35% Ammonium Sulfate Pellets. Previous
investigations with liver FPGS indicated a limited ability of the
enzyme to form longer chain length methotrexate polygluta
mates (Gluj and higher) (21, 22, 24, 25), as was the case with
cultured hepatocytes during short term incubations (14). Since
the rat hepatoma cells readily formed longer chain derivatives
under similar conditions (13,26), an evaluation of the products
formed with hepatoma cell extracts was conducted to determine
if the enzyme from this source could more readily produce 4NH2-10-CH3-Pte-Glu4and Glu5. Methotrexate at an extracel2428
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REGULATION BY FOLYLPOLYGLUTAMATE SYNTHETASE
lular concentration of 10 /¿-M
was extensively glu tam ylaied by
intact wild-type H35 hepatoma cells in 6 h (Table 4), with the
predominating products (73%) containing four or more gluta
mate residues. In contrast, FPGS from crude extracts synthe
sized polyglutamates from methotrexate only up to the triglutamate level (Table 5). Partial purification of the extracts by
ammonium sulfate fractionation did not result in a preparation
which could catalyze the formation of significant amounts of
the tetraglutamate (Table 6). This partially purified extract was
capable of converting 4-NH2-10-CH3-PteGlu3 to 4-NH2-10CH.i-Pte-Glu4, and the amount formed was highly dependent
on the amount of substrate (Table 6). The same reaction oc
curred in crude extracts, but at a 50-70% reduction amount
(data not shown).
The monoglutamate of the thymidylate synthase inhibitor
PDDF (10 MM)was also glutamylated by intact H3S hepatoma
cells (Table 4), with the glutamylated products consisting nearly
exclusively of the tetra- or higher polyglutamate derivatives. A
radiolabeled peak near the expected retention time for the
pentaglutamate derivative of PDDF was detected during the
high-performance
liquid chromatography
product analysis,
which increased when the incubation time was extended from
6 to 24 h. Lack of a pentaglutamate standard prevented positive
identification. In contrast to methotrexate, PDDF was readily
Table 4 Metabolism of methotrexate and PDDF in H35 hepatoma cells
H35 cells were grown as described in "Materials and Methods" for 96 h. The
culture medium was changed to folate-free Swims' S-77 medium plus insulin (10
mU/ml), and 24 h later the cells were incubated with [3H]methotrexate or ['V|
PDDF for the indicated time period. Polyglutamate formation was monitored as
described in "Materials and Methods." Data are presented as the mean of at least
two separate experiments. Glu, is indicative of all species of Glu«and greater.
Cellular polyglutamates
Compound
Concentration
OIM)
Incubation
(h)
Concentration
(nmol/g)
% Distribution
Glu2 (.lu,
Glu«
able to form substantial amounts of the tetra- and higher
polyglutamate derivatives when using crude extracts of folatedeplete H35 hepatoma cells as an enzyme source (Table 5).
Using a resuspended 0-35% ammonium sulfate pellet from
crude extracts as a source for FPGS (Table 6), the product
distribution formed after 6 h from PDDF at initial concentra
tions lower than 1 ^M was virtually identical to that seen in the
experiments with intact cells (i.e., near-exclusive formation of
the tetra- and higher polyglutamate derivatives).
DISCUSSION
Several detailed investigations using FPGS from normal
mammalian tissues have appeared in the recent literature (1720), but relatively few studies have used rapidly dividing or
transformed cells as an enzyme source. Taylor and Ilamia
showed that the FPGS activity of suspension-cultured Chinese
hamster ovary cells was not altered by the growth state of the
cells or the addition of several metabolites (glycine, adenosine,
thymidine, folates, and methionine) to the media (37), suggest
ing that changes in growth state and culture conditions did not
result in altered activities or amounts of the enzyme. However,
the finding that the ability of H35 hepatoma cells to glutamylate
methotrexate could be markedly altered by changes in growth
state, cellular folate levels, and the exclusion of methionine
from the media (14, 26) suggested the possibility that changes
in FPGS activity in cells could be involved in regulating the
glutamylation of methotrexate. This possibility has been tested
in the present study by measuring FPGS activity in extracts of
H35 hepatoma cells grown under those culture conditions
which caused the greatest alterations in whole cell glutamyla
tion.
Using crude extracts of H35 cells as a source for FPGS,
linear incorporation of [3H]glutamate into 250 MMmethotrexate
took place for up to 1 h with 400 /¿gof added protein, and up
to 2 h with 200 Mgof added protein (Table 1). These data, along
with the finding that -y-glutamyl hydrolase [optimum pH 7.2
(34)] was virtually inactive under these conditions (pH 8.4),
Table 5 Analysis of products formed by FPGS from crude extracts offolateindicated that a reliable comparison of FPGS activity could be
depleted H35 hepatoma cells using methotrexate and PDDF as substrates
Standard assay procedure as described in "Materials and Methods" was em
made by assaying the extracts in duplicate for 1 h at 400 /¿g
of
ployed using the indicated concentration of methotrexate or PDDF, a 6-h incu
added protein and for 2 h at 200 ¿tgof added protein. At
bation time, and 0.8 mg of added protein. A crude extract of confluent folate
saturating substrate concentrations, the 1.8-fold higher gluta
depleted H3S cells was used as an enzyme source.
mate incorporation for methotrexate versus tetrahydrofolate
% Polyglutamate
compared favorably with the results of similar experiments by
SubstrateMethotrexateMethotrexateMethotrexatePDDFPDDFPDDF(MM)1010.21010.2(pmol)8831113180817942Glu26358783210Glu,364222565839Glu«»100414151
McGuire et al. using partially purified rat liver FPGS, which
showed an approximate 1.6-fold greater glutamate incorpora
tion with methotrexate (21).
The results of these experiments suggest that the alterations
in the cellular glutamylation of methotrexate are in part a
consequence of changes in the activity of FPGS (Table 2). The
reduction of cellular folates (i.e., comparing control cultures of
Table 6 Analysis of products formed by FPGS from resuspended 0-35%
wild-type versus folate-deplete H35 cells), caused comparable
ammonium sulfate pellet of crude extracts of 1135 hepatoma cells
increases in both whole cell glutamylation and extract FPGS
Standard assay procedure as described in "Materials and Methods" was em
activity (approximately 1.8-fold), indicating that alterations in
ployed using a 6-h incubation time and 400 Mgof added protein.
the enzyme itself may be largely responsible for this effect.
Polyglutamate1Glui655859500800Glu,324241129059120Glu«*30083911003388100
However, media additions of folinic acid and 5-methyltetrahyConcentration
incorporatec
OIM)4-NHj-lO-CHj-PteGlu,4-NHj-lO-CHj-PteGlu,4-NH2-10-CH,-PteGlu,4-NH2-10-CH,-PteGlu,4-NHj-10-CH,-PteGlu,4-NH2-10-CH,-PteGlu,PDDF
Substrate
(pmol)10111381824140364316734% drofolate to wild-type and folate-deplete cultures for 24 h, which
caused a near-quantitative reduction in cellular methotrexate
glutamylation [Table 2, (26)], resulted in only a 50% decrease
in extract FPGS activity, demonstrating that these changes in
enzyme activity are probably not large enough to totally account
for alterations in glutamylation in intact cells. Thus, other
(Glu),PDDF
(Glu),PDDF
factors which could alter cellular glutamylation of the drug,
(Glu),1010.21001011010.2Glutamate
such as the competition between reduced folates and methoMethotrexatePDDFPDDF1010106624113.011.122.240023207398100
2429
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REGULATION BY FOLYLPOLYGLUTAMATE SYNTHETASE
trexate for transport (38) and FPGS (21, 22, 39), can also
import an additional effect.
The increased FPGS activity in extracts of H35 cells har
vested in log phase (Table 2 and Fig. 1) indicates that changes
in enzyme activity are also involved in the increased ability of
dividing cells to glutamylate methotrexate. These data are the
first of their kind and are in contrast to the results of similar
experiments by Taylor and Hanna (37), who reported that
FPGS activity of suspension-cultured Chinese hamster ovary
cells was not altered by growth state. A report of increased
FPGS activity in dividing LSI78Y lymphoma cells has recently
appeared in abbreviated form (40).
Methionine has been shown to alter both folylpolyglutamate
product distribution in Chinese hamster ovary cells (41) and
the rate of polyglutamylation in hepatic cells. In the latter case,
glutamylation was reduced by the presence of methionine in
normal hepatocytes and increased by the presence of methio
nine in folate-restricted hepatoma cells (14). FPGS activity in
extracts of H3S cells deprived of methionine was decreased
57% compared to extracts of control cultures (Table 3), while
the corresponding decrease in whole cell glutamylation was
70% (14). The combination of folinic acid addition and methi
onine exclusion caused the largest reduction in extract FPGS
activity measured during this study (>7S%). The mechanism by
which methionine alters FPGS activity is not understood, but
the effect is particularly interesting because of its magnitude
and divergent nature in hepatocytes and hepatoma cells. Ex
periments in which FPGS activity was measured in extracts of
hepatocytes indicated that the reduction in glutamylation is not
associated with changes in enzyme activity.
One possible additional mechanism for alterations in gluta
mylation is an alteration in the steady state intracellular levels
of methotrexate as a result of different conditions. Several
positive effectors of the rate of MTX glutamylation which
include insulin, methionine, and folate-Iacking medium (26)
caused no discernible alteration in the steady state levels of
methotrexate during the period of polyglutamate formation in
intact cells.4 Similarly the other substrates for FPGS (ATP and
glutamate) are not altered in amount by the changes in growth
conditions. However dividing hepatoma cells accumulated more
intracellular methotrexate than confluent cultures when incu
bated in the presence of a concentration of the drug (10 /IM)
that is saturating for transport and glutamylation. Under these
conditions confluent cells had 8.8 ±3.7 nmol/g and dividing
cells had 14.2 ±6.7 nmol/g methotrexate (N = 6). Thus, this
1.6-fold increase in cellular MTX may also contribute to the
enhanced rate of glutamylation observed in dividing cells.
Shane and his coworkers have conducted a series of elegant
studies in which purified FPGS from hog liver was used to
model folate and folate analogue metabolism, and concluded
that changes in cellular folylpolyglutamate synthetic rates and
distributions can be explained by substrate specificities and
affinities for the enzyme (25). Furthermore, Shane has pointed
out that an exception to autoregulatory control by substrate
specificity would exist in the case of substrates such as metho
trexate (and its polyglutamates), whose metabolism is relatively
slow. Under these conditions, modest changes in the level or
activity of FPGS would play an important role in the rate of
conversion to polyglutamates (25). Thus, the present finding
that extracts of H35 hepatoma cells with differential capacities
for glutamylation have correspondingly altered FPGS activities
suggests that the cellular levels or activity of this enzyme may
4 T. B. Johnson and J. Galivan, unpublished results.
play an important role in the regulation of methotrexate me
tabolism in transformed mammalian tissue. The activity of 7glutamyl hydrolase, the enzyme which catalyzes the hydrolysis
of folyl and antifolyl polyglutamates, does not appear to be a
major factor in altering glutamylation in intact H35 cells as a
result of growth state, folate, and methionine content, as its
activity in extracts of cells grown under these conditions is not
significantly changed.4
Long chain methotrexate polyglutamates (those with four or
more glutamate residues) can be formed in hepatoma cells as
early as 2 h after exposure to methotrexate and can be the
major species within 4 h (13, 26), while virtually no methotrex
ate species above the level of the triglutamate are formed in
extended (>36 h), enzyme-supplemented incubations with par
tially purified FPGS from rat and mouse liver (22, 24). The
latter is consistent with the reduced capacity of cultured hepa
tocytes to form longer chain length polyglutamates during
short-term incubations (<6 h). In this investigation, FPGS from
crude extracts synthesized polyglutamates from methotrexate
only up to the triglutamate level (Table 5), in contrast to
observations with intact cells (13, 26). The absence of higher
polyglutamate products was not a consequence of 7-glutamyl
hydrolase activity, since ammonium sulfate fractionation
[which greatly reduced the hydrolase activity in the extracts
(23)] failed to yield a preparation which could form significant
amounts of 4-NH2-10-CH3-Pte-Glu4 from methotrexate (Table
6). Additionally, the ammonium sulfate fraction was able to
form 4-NH2-10-CH3-Pte-Glu4 from 4-NH2-10-CH3-Pte-Glu3,
indicating that this conversion could take place provided the
triglutamate was present at effective concentrations. Thus in
spite of the fact that the hepatoma cells have a greater capacity
to form long chain polyglutamates of methotrexate relative to
hepatocytes, the FPGS extracted from both sources has a
limited capacity to convert the drug to its high polyglutamates.
In contrast to the result with methotrexate, both intact H35
hepatoma cells and FPGS from extracts metabolized the nionoglutamate of the thymidylate synthase inhibitor PDDF to long
chain polyglutamates (Tables 4 and 5). Furthermore, a similar
product distribution for PDDF as was seen in whole cell exper
iments was noted when using FPGS from crude extracts or
resuspended 0-35% ammonium sulfate pellets (Table 6). These
results suggest that the inability of methotrexate to form long
chain polyglutamates outside of the cellular environment is due
to the triglutamate being formed in quantities insufficient to
overcome its poor kinetic properties as a substrate for further
glutamylation. Shane et al. have shown that the diglutamate
was the highest chain length formed for several 4-amino unti
folates tested with the purified hog liver enzyme (25). Decreased
substrate activity of methotrexate polyglutamates as the gluta
mate chain length is increased has been reported by Schoo et
al., who showed that 4-NH2-10-CH3-Pte-Glu3 had about 3-5%
of the substrate activity of methotrexate for the beef liver
enzyme (22), and by Clarke and Waxman, who found that the
relative Vm^/Km ratios of 4-NH2-10-CH3-Pte-Glu3.5 for the
human liver enzyme were each less than 20% that of metho
trexate (42). It does not appear that the catalytic ability of
FPGS is severely compromised outside of the cell, since the use
of other substrates such as reduced folates (4, 17, 19, 23, 25)
and PDDF (Tables 5 and 6) can result in the formation of long
chain polyglutamates. Other possible factors contributing to
the limited glutamylation of methotrexate in experiments with
isolated enzyme may be the inability to achieve the effective
cellular concentration of FPGS, and the absence of cellular
proteins which may alter glutamylation.
2430
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REGULATION BY FOLYLPOLYGLUTAMATE SYNTHETASE
The results of this study show that the activity of FPGS may
be an important parameter in the regulation of methotrexate
polyglutamate formation in H35 hepatoma cells, and that the
decreased ability to form long chain methotrexate polyglutamates in assays with isolated enzyme may be a consequence of
the decreased substrate activity of the polyglutamates of this
antifolate, rather than solely an impairment of the enzyme
catalytic ability. The interaction of methotrexate and PDDF
with intact cells and FPGS may take on added importance since
it has recently been shown that this combination and others
like it can exhibit synergistic cytotoxicity against hepatoma
cells in vitro (43).
20.
21.
22.
23.
24.
25.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr. John J. McGuire for help
and advice with the FPGS assay and Zenia Nimec for her excellent
technical assistance.
27.
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Role of Folylpolyglutamate Synthetase in the Regulation of
Methotrexate Polyglutamate Formation in H35 Hepatoma Cells
Thomas B. Johnson, M. G. Nair and John Galivan
Cancer Res 1988;48:2426-2431.
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