[CANCER RESEARCH 38, 2989-2996, September 1978)
0008-5472/78/0038-0000$02.00
Effects of Methotrexate and of Environmental Factors on
Glycolysis and Metabolic Energy State In Cultured
Ehrlich Ascites Carcinoma Cells1
Edvardas Kaminskas2 and Ann C. Nussey
Department of Medicine, University of Wisconsin-Mount Sinai Medical Center, Milwaukee, Wisconsin 53201
ings prompted an examination of environmental factors
that affected cell multiplication, glycolysis, and the meta
Effects of nonpermissive environmental growth condi bolic energy stale in Ehrlich ascites cells grown in continu
tions and of methotrexate(amethopterin)(MIX) on rates ous cultures. Previous studies with EhrIich asciles tumor
of glycolysis, adenylate pools, and adenylate energy cells obtained from animals could not lake into account the
charge were examined in cultured Ehrlichascites card nutritional slate of cells and the influence of environmental
noma cells. Glycolytic rates were decreased in cells factors, which could be examined in cell culture. Usually,
incubated at 25°,at pH 6.2, In amino acid-free medium, cells obtained from animals had grown to very high-cell
with cydloheximide,actlnomycinD, and with MTX; were densities, were probably starved of amino acids (14), and
increased in cells incubated at 43°,at pH 8.4, and were possibly of glucose since the ascitic fluid may contain no
normal in serum-, phosphate-,or glucose-deprivedcells. detectable glucose (27).After collection the harvestedcells
Adenylatepoolswere decreasedIn cells Incubatedat 25°, were suspendedat extremely high densities in buffers some
at pH 6.2, pH 8.4, In amino acid-free, in glucose-free,in of which, such as Tris, have been shown to be toxic to
MTX-contalning media, and were normal in serum-dc some cultured cells (6). In this study cells were grown under
prived cells and in cells Incubated at 43°.Adenylate conditions that permitted maximal ratesof protein synthesis
energy chargewas decreased In glucose-deprivedand in and cell multiplication as described previously (19, 21). Cell
MTX-treated cells and was elevated In serum-deprived doubling limes were as rapid as during the exponential
cells and In cells Incubatedat 25°.
MTX at growth-inhibit growth phase in the animal (22). Cell growth was arrested
lng concentrationsproduced an Inhibitionof glycolysis by deletion of specific nutrients or by the culturing of cells
that was very severe in low-glucose media and less under nonpermissive thermal or pH conditions. In fibro
severe in high-glucosemedia.Glucoseconsumptionrates blastic cells rates of sugar uptake and of glycolysiS were
and cell survivalincreasedwhen cells were shiftedfrom found to be dependent on cell density, growth rate, pH,
low-glucoseto high-glucosemedia. Decreases In ade temperature, glucose starvation, change of medium, and
nylate poolswere more pronouncedin low-glucosethan additions of serum and of insulin (12, 30); initiation of cell
In high-glucosemedia. Hypoxanthineand thymidinepre growth was correlatedwith increasedratesof sugar uptake
vented MTX cytotoxiclty.Hypoxanthinepreventedthe de (3, 32, 33, 35).
crease in adenylatepoolsand the inhibitionof glycolysis;
The second part of this study examines the effects of
thymidlnewas less effective. Decreases in cell viability MTX3on glycolysisand on the energystateof cellsin order
were correlated with severe Inhibitionof glucose con to evaluatethe contribution of theseeffects to MTXcylotox
sumptionand with adenosine 5'-triphosphatelevels de icily. MTX has been described as causing “purineless
creased to less than 10% of normal. A major cytotoxic death―
by inhibiting DNA, RNA,and protein syntheses(15).
effect of MTX appears to be due to an energy-depleted MTX also inhibits thymidylatesynthesisand thus DNA
state.
synthesis, resulting in “Ihymineless
death―
(5). The relative
importance of each cytotoxic mechanism has not been
INTRODUCTION
defined for most tumors or tumor cell lines.
ABSTRACT
Ehrlich ascites carcinoma cells in culture are dependent MATERIALS AND METHODS
on glycolysis for generation of metabolic energy in the form
of ATP. The evidence for this view is that deprivation of
Materials. Powderedminimalessentialmedium(Eagle's)
glucose rapidly leads to a decreased ATP/ADP ratio, mdi with Earle's salts and FCS were purchased from Grand
cating a defect in ATP regeneration and to a decreased Island Biological Co., Grand Island, N. Y. Morpholinopro
adenylate energy charge (ATP + @/2
ADP/ATP + ADP + panesulfonic acid, glulamine, hypoxanthmne,MTX, and
AMP) (24), indicatinga suppressionof ATP-utilizingreac dThd were purchased from Sigma Chemical Co., St. Louis,
lions and an activation of ATP-generating reactions (1). In Mo.; ATP, ADP, and AMP from P-L Biochemicals,Inc.,
contrast inhibition of respiration in Ehrlich ascites tumor Milwaukee, Wis.) and sodium dodecyl sulfate from Bio-Rad,
cells in the presence of glucose does not result in such Richmond, Calif. GOD-PERID(trade name) and lactate test
changes of metabolic energy parameters (39). These find
kits were purchased from Boehringer-Mannheim Corp.,
NewYork, N. Y. Freshsolutionsof MTX, hypoxanthine,and
I
Supported
by
Mount
Sinai
Medical
Center-University
Affiliation Program.
I To whom requestsfor reprintsshould be addressed.
SEPTEMBER
of
Wisconsin
3 The
abbreviations
used
are:
MTX,
methotrexate
(amethopterin);
FCS,
fetal calf serum; dThd, thymidine.
1978
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
2989
E. Kaminskas and A. C. Nussey
dThd were made for each experiment and kept at —20°
until cell pellets were washed once with 1.0 ml of phosphate
shortly before use.
buffered saline, solubilized with 1%sodium dodecyl sulfate,
Cultivationof Cells. Ehrlichascilescarcinomacellswere and their absorbance at 260 nm and the protein content
grown in continuous cultures in spinner flasks in a 37° were determined (25). The absorbance at 260 nm of cell
incubator. Cells were passaged in random-bred Swiss lysateswas found to be an accurate measureof cell number
while mice every 6 to 12 months, and stocks were kept at (21); in 100 determinations in triplicate, single determina
tions deviated from the mean by an average of 3.5%. A
frozen stocks. The growth medium was minimal essential lysale of 9.6 x 10@exponentially growing cells had an
medium (Eagle's) with NaHCO3content reduced to 13 mM, absorbance of 1 A2@unit and contained 0.24 mg of protein.
and supplemented with 1 mg/mI of glucose, 25 mM mor Glucose consumption rates were calculated as nmol of
pholinopropanesulfonic acid, 2.5% heat-inactivated FCS, glucose consumed per mm per mg of cell protein. Con
penicillin G (75 units/mI), and slreptomycin (50 @g/ml);
pH sumplion rates were not computed for experiments in
of the medium was adjusted to 7.3 with NaOH. The cell which cells consumed less than 0.2 mg/mI of glucose. For
concentration was kept in the range of 2 to 12 x 10@cells/ comparison of the rates obtained for cells in parallel cul
ml by daily dilution with fresh medium. Cells were resus lures, only linear portions of glucose depletion curves at
pended in fresh medium at least twice weekly. Glucose-, comparable glucose concentrations were used. Lactate
phosphate-, and amino-acid-deficient media were prepared productionrateswere computed as nmol of lactatepro
by omitting each of these components, respectively, and duced per mm per mg of cell protein.
adding 2.5% dialyzed FCS. FCS was dialyzed against 100
AdenylateConcentrations.Cells(1 x 10@)
werecollected
(at 25°
and at 4°
volumes of double distilled, demineralized waler for 24 hr by cenlrifugation at 200 x g for 5 mm at 37°
and filtered through a 0.20 @mfiller; it contained no for cells incubated at those temperatures) and extracted
delectable glucose. During routine cultivation cells were with cold 0.7 NHCIO4with vigorous mixing. After 1 or more
not subjected to inadvertent glucose starvation, since the hr at 4°,the extract was centrifuged at 10,000 x g for 5 mm
growth medium after 24 hr of cell growth still contained at at 4°.The supernate was brought up to pH 6.5 with 10 N
least 0.5 mg/mI glucose. Before being used for experi KOH. The resultant KCIO4precipitate was sedimented by
ments, cells were resuspended in fresh medium and incu centrifugalion at 10,000 x g for 10 mm at 4°.
The volume of
bated for at least 2 hr. The experimental cultures were the supernale was recorded and adenylale concentrations
incubated in water baths equipped with B. Braun Thermo were measured by thin-layer chromatography as previously
mix II healer-circulators. Measurements of pH were per described (24). MTX appears to produce quenching of the
formed with a Radiometer Model 62 pH meter at room nucleotide spots when the thin-layer polyelhyleneimine
temperature. The viability of cells, as judged by trypan blue cellulose sheets are examined under a UV light. However,
exclusion, was over 95% under all experimental conditions this did not interfere with measurements of adenylates,
except at 42-43°,in media containing MTX and after 19-hr since ATP, ADP, and AMP added to extracts of MTX-treated
incubations
inglucose-free
medium. Cellcountsingrowth cells were completely recovered in eluates of spots with
experiments were performed with a hemacytometer in typical migration characteristics of these nucleotides. A
quadruplicate. All cell concentration data are presented as sheet spotted with standard solutions of ATP, ADP, and
viable cell counts. II was found that when cultures, which AMPwasrunin parallelwiththe sheetsontowhichextracts
contained a significant proportion of cells staining with of normal and MTX-lrealed cells were spotted. Standard
and
trypan blue, were centrifuged and resuspended, over 97% solutions of adenine nucleotides were kept at —170°,
excluded trypan blue. Thus the nonviable cells lysedduring were thawed shortly before use.
this procedure.
Rates of GlucoseConsumptionand of Lactate Produc RESULTS
tion. The ratesof disappearanceof glucosefrom the me
dium and of appearanceof lactate in the medium were used
Glycolysis, Adenylate Pools, and Adenylate Energy
to measurethe rates of glucose consumption and of lactate Charge in Cells CultivatedunderGrowth-inhibitingCondi
production by cells. For this purpose cells were incubated tions. Ehrlich asciles carcinomacells consumedglucose
in complete growth medium containing 1 mg/mI (5.5 mM) from the medium in a linear fashion with time until glucose
of glucose, or other concentrations as specified at cell concentration in the medium decreasedto lessthan 1.5 mM
concentrations between 3.3 x 10@and 5.7 x 10@cells/mI (Chart 1).This linearity of glucose consumption was present
for 2 to 6 hr. At hourly intervals 1.0 ml samples of cultures under all experimental conditions described below, except
were removed with an Eppendorf pipel for determinations when cells were incubated with inhibitors of RNA and pro
of pH, glucose and lactate concentrations, and of cell tein syntheses, and with MTX. As cells consumed glucose
density. The sampleswere centrifuged at 300 x g for 3 mm from the medium, the adenylale pools in cells decreased
at 18°.
Glucose concentrations in the medium sampleswere but the adenylate energy charge remained unchanged.
determined in duplicate by the glucose oxidase method, Compared to cells harvested from 6 mM glucose medium,
glucose medium in parallel
with the GOD-PERIDtest kit (2); in 120 determinations cells harvested from 1.5 mp@i
duplicate determinations deviated from the mean by an cultures had adenylale pools decreased by about 33%.
average of ±1.5% (S.E.). Lactate determinations by the Cells excreted 1.6 to 1.8 mol of lactate per mol of glucose
lactate dehydrogenase method (9) were usually single be consumed, except during incubations in the pH range of
cause of the sensitivity of the assay; in duplicate determi 8.0 to 8.5, when the lactate/glucose ratio was 1.1 to 1.4.
Cells incubated at 37°and in the pH range of 7.1 to 7.5
nations standard error was the same as for glucose. The
— 1 70°.
2990
Fresh
cultures
were
started
every
3
to
4
months
from
CANCER RESEARCH VOL. 38
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
MTX Effect on G!ycolysis and Energy State
Table1
Glucoseconsumption rates, adenylatepools, and adenylate
energycharge in growth-inhibited cells
Glucose
consump
E
Medium a
1. 37°,pH7.3
2. 25°,4hr
3. 43°,4hr
4. pH 6.2, 20 hr
5. pH 8.4, 2Ohr
0
2
4
6
24
HOURS
Chart 1. Inhibition of glucoseconsumptionby 2 x 10' PAMTX.Cellswere
Incubated at 3 x 10. cells/mI for 1 hr in serum-free medium containing icrx,
6. Minusserum,20hr
100
26± 3
148± 8
13± 2
.
100
84±15
96±4
Energy
charged
- 0.84 ±o.06@
0.94±0.04
0.82±0.05
76±10
89± 3
0.78 ±0.08
0.77 ±0.08
0.85 ±0.03
92± 3
85±5
0.45 ±0.05
66±15
76±
0.75±0.10
65±15
95± 5
254 ±42
104 ±20
56±
5
Minus serum, 20 hr;
Plusserum,2 hr
7. Minus glucose, 1 hr
Minusglucose,19hr
then were suspendedat 3 x 10' cells/mI in 2.5% dialyzed FCS medium
without MTX (C) and in the samemedium supplementedwith 1 x 10@M
8. Minusphosphate,
48
hypoxanthine (1k). Another culture was not treated with MTX and was
incubated in parallel ( 0). Samples of culture were taken at times as indicated
9. Minus amino acids, 1
for determinationsof glucosecontentand of cell density.
Adenylate
tion rate@@ pooIs@' (as
(as % of
% of con
control)
trol)
113 ±30
95 ±10
0.92±0.02
100± 1
104± 7
hr
hr
10. PIus cycloheximide
5
0.85±0.04
(10 @g/ml),4 hr
doubled in number every 12to 15 hr (19).Cells incubated at 11. Plusactinomycin
D
75 ± 5! 95 ± 5
0.86 ±0.03
25°remained at a constant density over a period of several
(0.4 @g/ml),4 hr
days; cells incubated at 42-43°declined progressively in
a Cells were incubated in experimental media as shown and in
number, Glucose consumption and lactate production normalmediumin parallelcultures;glucoseconsumptionrates
rates, as tested in parallel cultures, were found to increase and adenylate pools were determined in 3 to 9 experiments of each
exponentially between 25°and 40°with a Q10of 2 (not type.
b Glucose
consumption
rates in control cultures
were 20 ± 5
illustrated). Cells incubated at 25°and at 43°consumed nmol/min/mg of cell protein in 90% of determinations. Rates in
glucose at 26% and at 148% of the rates at 37°(Table 1). experimental cultures are expressed as percentage of the rates in
The adenylate pools and adenylate energy charge were control cultures incubated in parallel.
pools
in
control
cultures
were
6.3
± 0.6
nmol/10@
normal in cells incubated at 43°.In cells incubated at 25° C Adenylate
Adenylatepools in experimentalcells are expressedas
and at 4°,the adenylate pools were slightly decreased and cells.
percentage of the pools in control cells incubated in parallel
the adenylale energy charge was increased (Table 1). This cultures.The data in Ref.24 on adenylatepools and adenylate
increase in the energy charge was due to preferential energychargein serum-,glucose-,andaminoacid-deprivedcells
excretion of AMP and of ADP as compared to ATP into the are includedtogetherwith additionaldeterminationsunderthese
medium (E. Kaminskas,unpublished data). Cell growth was conditions.
d Energy charge was calculated
as M ratio of ATP + 1 /2 ADP/
effectively arrested in media at pH 6.1 to 6.3 and at pH 8.2 ATP+
ADP+ AMP.
to 8.5 without loss of cell viability, as measuredover a 3 day
e Mean ±SE.
period. Cells had to be incubated at concentrations of
I Glucose consumption rates were normal during the first 2 hr
about 1 x 10@cells/mI in pH 8.2 to 8.5 media; cells anddecreasedin subsequenthr asshown.
incubated at higher densities decreased the pH of the
incubated in complete medium in parallel cultures (Table
medium and proceededtogrow rapidly.
Glucoseconsump
lion rates of cells incubated at pH 6.2 and at pH 8.5 were 13 1). Supplementation of the amino acid-free medium with
and 254%,respectively, of the ratesof cells incubated at pH glutamine or with 1 x 10@ M hypoxanthine resulted in
7.3 (Table 1). In other experiments we found that glucose increased glucose consumption rates in some experiments
consumption rates increased as a linear function of the but not in others. Addition of cycloheximide (10 @g/ml),
medium pH in the pH range of 6.1 to 8.5 (20).The adenylate which inhibited labeled amino acid incorporation by over
pools were decreased by 44 and 24% in cells incubated at 95% and labeled adenine incorporation by 35%, decreased
pH 6.2 and at pH 8.4, respectively; the adenylate energy glucose consumption by 20 to 47%(Table 1). Cells that had
been incubated with cycloheximide for 24 hr consumed
charges remained normal (Table 1).
Cells subjected to serum, phosphate, or glucose starva glucose from 11 and 5.5 mMglucose media at about 60%of
lion had normal rates of glycolysis (Table 1). Metabolic the rates of untreated cells, as tested in parallel cultures.
shiftdown was documented by low-protein synthesis rates Addition of actinomycin D (0.4 @tg/ml),which inhibited
in cells deprived of serum for 20 hr. phosphate for 48 hr, adenine incorporation by over 93%, did not affect glucose
and of glucose for 1 and 19 hr. Reversalof serum starvation consumption in the first 2 hr and decreased it by 30% in the
by dialyzed serum was similarly documented (18). Since subsequent 5 hr. Addition of both inhibitors did not de
prolonged glucose starvation induced in fibroblasts an crease glucose consumption rates more than did the addi
increase in sugar uptake capacity (11, 17, 23, 26, 36), lion of cycloheximide alone. Adenylate energy charge was
Ehrlich asciles cells were cultivated in a glucose-free me increased in serum-starvedcells, normal in cells incubated
dium for 19 hr. An increase in glucoseconsumption rate with cycloheximide or with actinomycin D, slightly de
was not detected. Cells incubated in amino acid-free me creased in amino acid-starved cells, and markedly de
dium consumed glucose at 20 to 50%lower rates than cells creased in glucose-starved cells (24) (Table 1).
SEPTEMBER
1978
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
2991
E. Kaminskas and A. C. Nussey
Inhibitionof Glycolysisby MTX. Glycolysiswas inhibited incubated without MTX. This difference is an underestimate
of the MTX effect, since cells incubated without MTX had
x 10@to 5 x 10@M MTX (a concentrationrangeat which consumed essentially all glucose from the medium. When
cell multiplication was inhibited). In a typical experiment cells, which had been incubated for 24 hr in 2.9 mMglucose
(Chart 1) glucose consumption rate becamevery low after 4 medium with MTX, were resuspended in 11 mM glucose
hr of incubation and very little additional glucose was medium with MTXthey consumed glucose at the same rate
consumed over the subsequent 20 hr. As glucose consump as cells that had been continuously incubated in 11 mM
lion rates decreased, lactate production rates decreased in glucose medium with MTX (Table 2). This increase in
a disproportionate manner. In the first 2 to 3 hr the molar glucose consumption was reproducibly accompanied by an
in a marked and unusual manner in cells incubated with 5
lactate/glucose
ratio was 1.6 to 1.8, in the subsequent 3 hr
the ratio decreased to 1.1 to 1.3, and remained unchanged
after 24 hr. When cells, which were incubated for 24 hr with
MTX, were resuspendedin fresh 6 mM glucose medium
increased survival of cells (Chart 3). The data in Chart 3
Table 2
Glucoseconsumption rates of cells incubated with MTX
with MTX they consumed glucose in the same pattern. The
effect of glucose concentration on glucose consumption in
MTX-trealed cells is shown in Chart 2. Cells incubated in 12
GlucoseGlucose
concentra
rate@@DayAdditions4'tion
without
MTX, and this
rate was undiminished 3.5 hr after the addition of MTX.
Cells incubated in 6 and 3 mM glucose media with MTX
consumed 40 and 64% less glucose, respectively, than did
cells in 12 mM glucose medium with MTX. The effect of
lion
(mM)(p.mol/24
mMglucosemediumwith MTX consumedglucoseat about
60% of the rate of cells incubated
consump
hr1None
hr/10@
hr
cells)0
24
11.0
6.00
2.75
MTX
None
MTX11.0
0.202None
glucose concentration was even more evident when cells
were incubated at low densities for 24 hr. Cells incubated in
I 1 mMglucose medium with MTXconsumed glucose at the
2.92
2.92
11.0
MTX
None
ments (Table 2) and 3O%less in others. In contrast cells
incubated in a 2.9 mM glucose medium with MTX-con
sumed glucose at about 25 to 30% of the rate of cells
7.10
2.92
MTX
MTXC
MTX+
same rates as cells incubated without MTX in some experi
2.92
11.0
2.92
Hypoxan
thine+
1.60
0.67
0.06
2.421.41
3.03
1.17
0.22
0.67
2.64
6.99
2.591.30
0.16
1.50
0.12
dThd d11.0
a Cells were incubated with and without 2 x 10@ N MTX in 11
mM and 2.92 mM glucose media for 2 days at cell concentrations
(viable cell counts) as shown in Chart 3. Cells were resuspended in
fresh media after 24 hr. Glucose concentrations in the media were
determined atthe startof incubationsand after24 hr.
b Glucose
consumed
consumption
rate was calculated
in 24 hr by the mean concentration
as
@molof glucose
of cells (densities
of
viable cells at the start and at the end of incubation divided by 2).
C Cells
that
had
incubated
for
24
hr
in
2.92
mM
glucose
medium
with MTXwereincubatedin 11mMglucosemediumwith MTX.
d Cells which had incubated
for 24 hr in 2.92 M glucose
medium
with MTXwereincubatedin mediumcontaining2.92mMglucose,
MTX, 1 x 10-i N hypoxanthine,
and 1 x 10-i M dThd.
E
K
E
0
11
2
DAYS
Chart 3. Inhibition of cell multiplication by 2 x 10' N MTX as a function
0
1
2
3
HOURS
Chart2. Inhibition of glucose consumption by 2 x 10 N MT@as a
function of glucose concentration. Cells were incubated In parallel cultures
at 2.5 x 10•
cells/mI in serum-freemedia with (C) and without MTX (0)
containing glucose at concentrations as shown. Samples of cuftures were
takenat timesas indicatedfor determinationsof glucosecontentand of cell
density.
2992
of glucose concentration of the medium. Cells were suspendeddaily at
concentrations as shown in 2.5% dialyzed FCS media containing no glucose
(a), 2.92mMglucose(0), 11mMglucose(0), 2.92mMglucoseplusMTX
(•),
and11mM glucose
plusMTX (@)andincubated
inparallel
cultures.
One portion of the culture, which had incubated for 1 day in 2.92 [email protected]
plus MTX medium, was then incubated in 11 mM glucose plus MTX medium
(V),and anothersuch portionwas incubatedin 2.92 mMglucose + MT@
mediumsupplementedwith 1 x 10@N hypoxanthineand 1 x 10' N dThd
(A).Concentrations
of viablecellsareshown.
CANCERRESEARCH
VOL. 38
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MTX Effect on Glycolysis and Energy State
@
are presented as numbers of viable cells. Percentages of
viabililies were 98 to 100% in untreated cultures, 96 and
71% in cultures in 11 mM glucose media with MTX, and 91
and 52% in cultures in 2.9 mM glucose media with MTX
after 24 and 48 hr, respectively. In cells shifted from 2.9 mM
to 11 mM glucose media with MTX and in the culture
supplemented with dThd and hypoxanthine, percentagesof
viabilities were 78 and 74%, respectively. Larger differences
in percentages of viabilities between 11 mM and 3 mM
glucose media with MTX were noted in other experiments;
‘g
in 1 they were 80 and 37% in 11 mM medium and 72 and 3%
in 3 mM medium after 24 and 48 hr, respectively. Glucose
consumption rates, decreases in densities of viable cells,
and in percentagesof viabilities were higher for cells incu
bated in 5.5 mM glucose medium with MTX than for cells
incubated in 2.75 mM glucose medium with MTX and lower
than for cells in I 1 mM glucose medium with MTX. When
cells were incubated in glucose-free medium, the concen
tration of viable cells decreased by 50 to 55% during each
24 hr period (Chart 3). Addition of MTXto the glucose-free
medium did not further enhance the rate of decrease in the
concentration of viable cells (not illustrated).
Inhibition of glucose consumption by MTX did not de
pend on the continued presence of MTX in the medium,
since cells incubated for 1 hr with MTX and then resus
pended in MTX-free medium were inhibited to the same
degree as cells incubated continuously in MTX-conlaining
medium for 24 hr. Undialyzedserum and, to a lesserextent,
dialyzed serum decreased the inhibition of glucose con
sumption by MTX. This protective effect of serum was
evident only in the first several hr of incubation; glucose
consumption was inhibited to about the same extent in
cells incubated with MTX for 24 hr in dialyzed FCS,5.5 mM
glucose medium, and in serum-free medium in parallel
cultures. Inhibition of cell growth by 2 x 10@M MTX was
the same in dialyzed and undialyzed FCS media (not illus
Irated). Cells declined in number by 70% during a 24 hr
incubation with MTX in serum-free medium, whereas cells
incubated in the same medium without MTX increased in
number to a variable extent (19).
Hypoxanthine (1 x 10-i M) added together with 2 x 10
M MTX
consistently
prevented
the
inhibition
of glucose
consumption, as shown in Chart 1. Cells incubated with 1
x 10-s M to 1 x 10@M dThdlogelherwith 2 x 10@M MTX
consumed glucose at a slightly higher rate than did cells
incubated with MTXalone, but the effect of dThd was never
as pronounced as the effect of hypoxanthine. Addition of
serine and glycine in the form of the nonessential amino
acid solution of Eagle's medium had no effect. The respec
live effects of hypoxanthine, dThd, and of both metaboliles
in decreasing the cytotoxicity of MTX are apparent in
changes of viable cell counts as shown in Chart 4. Cells
incubated with MTX, hypoxanthine, and dThd multiplied
almost as rapidly as cells incubated in normal medium
especially during the first 2 days of incubation. Supplemen
tation of MTX medium with either hypoxanthine or dThd
permitted lower rates of multiplication. Percentage viabili
ties were 98 to 100% in all cultures after 24 hr; after 48 hr
they were 89, 93, 87, and 95%, and after 72 hr, 69, 76, 88,
and 71% in cultures incubated with MTX, MTX + hypoxan
Ihine, MTX + dThd, and MTX + hypoxanthine + dThd,
SEPTEMBER
DAYS
Chart4. Effectsof 1 x 10@N hypoxanthineand of 1 x 10' N dThd on
cell multiplication
in 2 x 10'
N MTx-containlng
medium. Cells were
suspendeddaily at concentrationsas shown in 2.5%dialyzed FCSmedia
containingno additions(0), MDC(s), MT@+ dThd (A), MTX+ hypoxanthine
(tx), and MT@+ dThd and hypoxanthine (0) and incubated In parallel
cultures. Concentrations of viable cells are shown.
respectively. Additions of both hypoxanthine and dThd to
cells, which had incubated for 24 hr in 2.9 mM glucose
medium with MTX, were far less effective in counteracting
the cytotoxicity of MTX than when the metaboliles were
added together with MTX at the start of incubation (Charts
3 and 4). II should be noted that shifting of cells from low
glucose medium to high-glucose
medium increased the
survival of cells to about the sameextent as the additions of
hypoxanthine and dThd to the low-glucose medium (Chart
3). Interestingly, glucose consumption of these cells re
mained depressed(Table2).
Table 3 shows representative experiments on the ade
nylate levels in MTX-treatedcells. After 3, 24, 48, and 72 hr
incubations with MTXin 11 mMglucose medium, ATP levels
in cells were reduced by 60, 88, 92, and 92%, respectively
(Table 3). After 48 hr the glucose consumption rate de
creased by 90%, as did cell viability. In cells incubated with
MTX in 2.75 mM glucose medium, ATP levels were de
creased by 72 and 96% after 24 and 48 hr. respectively.
Adenylale energy charge was decreasedafter 24 hr in both
media (Table 3). Addition of hypoxanthine together with
MTX preventedthe decreasesin ATPconcentrationsand in
the adenylate energy charge (Table 3). Addition of dThd
together with MTX resulted in lesser decreases in the ATP
levels and in the adenylate energy charge. When hypoxan
thine was added for 3 hr to cells, which had been incubated
with MTX for 24 hr, there was only a slight increase in the
concentration of ATP.
DISCUSSION
Rates of glycolysis in cells inhibited by environmental
conditions reflected the specific effects of these conditions
on the glycolylic pathway. Glycolytic rates did not correlate
with the metabolic energy state of cells as estimated by the
adenylale energy charge. The effects of temperature on
glycolytic rates were most likely due to the temperature
dependence characteristics of glucose transport and of
glycolytic enzymes. The temperature dependence of 2-
1978
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
2993
E. Kaminskas and A. C. Nussey
Table 3
Adenylateconcentrations in cells incubated with MTX
ATP + ADP +
AMP
Experi
(nmol/
ment
Time (hr)
Addition
1―
None
2'@
MTX
None
3
3
24
24
MTX
MTX
MTX
3c
48
72
24
24
None
MTX
MTX
4d
% (+1
(+1
106charge5.911001000.772.9750400.676.891001000.821.7926120.481.211880.440.741180.66
cells)% —MTX)ATP —MTX)Energy
48
None
24
MTX
MTX +
24
24
Hypoxan
thine
MTX + dThd
MTX +
24
24
3.12
6.44
49
100
40
103
0.65
0.76
24+3
2.30
36
18
0.51
Hypoxan
thine +
dThd
MTX;MTX+
Hypoxan
thine
a Cells wereincubated at 4 x 10@cells/mI in parallel cultures in serum-free, 11 mM
glucose medium with or without 2 x 10@M MTX for 3 hr before extraction.
b Cells were
incubated
daily in parallel
cultures
in fresh
1 1 m@ glucose,
2.5% dialyzed
FCS media with or without 2 x 10@M, MTX at a starting density each day of 4 x 10@cells/
ml. Cell extracts were madefrom 1 x 10 viable cells at times as indicated.
C Procedure
as
in
b except
that
medium
contained
2.75
mM
glucose
and
the
density
of
cells at the beginning of each 24-hr incubation was 2 x 10@cells/mI.
d Cells
parallel
were
cultures
incubated
containing
in 11 mM glucose,
no additions,
2.5%
2 x
dialyzed
FCS
10@ M MTX,
medium
MTX
+
for 24 hr in
1 x
10@ N
hypoxanthine, MTX + 1 x 10@ M dThd, and MTX plus hypoxanthine plus dThd. The
density of cells at the start of incubation was 2 x 10@cells/mi. To 1 portion of culture
incubated
with MTX for 24 hr, hypoxanthine
was added for 3 hr before the cell extract
wasmade.
deoxyglucose uptake in fibroblasts (12, 30) and in Novikoff
rat hepatoma cells (31)exhibited similarO,0values as found
in the present study. Dependence of glycolysis on pH has
been thought to be due to pH dependence of glycolytic
enzymesand of phosphofructokinase in particular (10, 37).
We recently found that sugar transport in Ehrlich asciles
cells is also pH dependent and may be the single most
important factor in the pH dependence of glycolysis (20).
The decreased glucose consumption rates in amino acid
free medium were probably due to severe inhibition of
protein and RNA syntheses. The results of experiments with
cells deprived of serum, phosphate, or glucose suggest that
growing and growth-arrested Ehrlich asciles cells consume
glucose at the same rate and that the stale of growth does
not affect glucose consumption capacity or requirement.
This conclusion suggests that the contrary results obtained
with fibroblastic cells may not be applicable to highly
malignant carcinoma cells. We drew a similar conclusion
from experiments that tested the role of cyclic AMP in the
growth control of Ehrlich ascites cells (21). The adenylate
pools were decreased in cells incubated in low-glucose
medium, inamino acid-free
medium, at25°,
inmedia atpH
creased. Thus growth inhibition due to a compromised
metabolic energy state could be postulated only in the case
of glucose starvation.
MTX inhibitedglycolysisin a mannernot encounteredin
studying the effects of environmental factors. The degree
of inhibition was very marked, particularly in media contain
ing physiological or subphysiological concentrations of
glucose. Decreased inhibition in the presence of dialyzed
serum may be explained by the binding of MTX by albumin
and the consequently reduced uptake by cells (8) and in the
presence of undialyzed serum by its content of nucleosides
(28). The inhibition of glycolysis by MTX is most likely due
to reduced ATP levels in MTX-lrealed cells, since the
addition of hypoxanthine prevented both the decrease in
ATP levels and the inhibition of glucose consumption. The
adenylale energy charge was reduced in MTX-trealed cells,
that in spite of adequateglucose levels in the culture media
the inhibition of glucose utilization by MTX resulted in a
limitation of ATP regeneration. The effect of glucose con
centration on glycolylic rates by MTX-lreated cells would
suggest a higher apparent K,,,for glucose. However, the
markedly decreased levels of ATP, the cosubstrale of glu
6.2 and at pH 8.4, and in glucose-free medium, but only in cose in the hexokinase reaction, and of fructose 6-phos
the last condition was the adenylate energy charge de phate in the phosphofruclokinase reaction probably ac
2994
CANCER RESEARCHVOL. 38
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
MTX Effect on Glycolysis and Energy State
count for this phenomenon. The increased rates of gly
colysis in high-glucose media are probably due to the mass
action effect of glucose influx into cells (Ref. 31; Fig. 10).
Glucose concentration in the medium also affected the
adenylale concentrations in MTX-lreated cells. Cells incu
baled with MTX in low-glucose medium had even more
(ad.),Methodsof EnzymaticAnalysis,Ed. 2, Vol. 3, pp. 1215-1222.New
York: Academic Press, Inc., 1974.
3. Bissel,M.J., Hatie,C., and Rubin,H. Patternsof GlucoseMetabolismin
Normal and Virus-transformed Chick Cells in Tissue Culture. J. NatI.
CancerInst.,49: 555-565,1972.
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Sd., 186:359-362,1971.
5. Caplzzi, R. L., Summers, W. P., and Bertino, J. R. L-Asparaginase
Induced Alteration of Amethopterin (Methotrexate) Activity in Mouse
reduced ATP levels than the very profoundly reduced levels
found in cells incubated in high-glucose medium. Inhibi
LeukemiaL5178Y.Ann. N. V. Aced.Sci., 186:302-311,1971.
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174: 501-503, 1971.
7. Freudenberg, H.. and Mager, J. Studies on the Mechanism of the
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Biochim. Biophys. Acts, 232: 537-555, 1971.
plelely explain the inhibition of glycolysis, since the inhibi
tion by cycloheximide and aclinomycin D was less pro
nounced and not dependent on glucose concentration.
Inhibition of glycolysis by MTX in extracts of Cloudman 591
melanoma cells was described by Hochstein (13). The
extent of inhibition was 10 to 25%, its mechanisms ap
peared to be an inhibition of the hexokinase reaction, and
addition of high levels of ATP reversed the inhibition (38).
The relative roles of inhibition of Ihymidylale synthesis
and of de noVo purine biosynthesis in the cytotoxic action
of MTX appear to vary in different tumor cell lines (4, 15,
16).Addition of purines to the medium reduced the cytolox
icity of MTX in L5178Y leukemia cells (16), but enhanced it
in L5178Y cells (4). Addition of both purines and dThd
reduced MTX cytotoxicity in L5178Y cells (4) and also in
Ehrlich ascites cells (Chart 4). Hypoxanlhine was more
effective than dThd in decreasing MTX cytoloxicity (Chart
4), and in this sense these results support Hryniuk's hy
pothesis that a ‘
‘purinelessslate―induced by MTX plays an
important role in MTX cytotoxicily (15). The results also
show that the “purineless
stale―is accompanied by a
severe inhibition of ATP regeneration and suggest that
“purineless
death―is due to an energy-depleted slate.
The present results were obtained with tumor cells highly
8. Goldman, I. D. The Characteristicsof the Membrane Transport of
Amethopterin and the Naturally Occurring Folates. Ann. N. V. Aced.
Sci.,
186:400-422,1971.
9. Gutmann,I., and Wahlefeld,A. W. L-LactateDeterminationwith Lactate
Dehydrogenase and NAD. Methods Enzymatic Anal., 3: 1464-1468, 1974.
10. Halpenn, M. L., Connors, H. P., Relman,A. S., and Karnofsky,M. D.
Factors that Control the Effect of pH on Glycolysis in Leukocytes. J.
Biol. Chem., 244: 381-390, 1969.
11. Hatanaka,M. SugarEffectsof MurineSarcomaVirus. Proc. NatI.Aced.
Sd. U. S., 70: 1364-1367, 1973.
12. Hatanaka,M. Transportof Sugarsin Tumor Cell Membranes.Biochim.
Biophys. Acts, 355: 77-104, 1974.
13. Hochstein, P. Synergistic Effects of 6-Mercaptopurine and Methotrexate
on AnaerobicGlycolysisby SubcellularFractionsof 591 Mouse Mela
noma.Proc.Am.Assoc.CancerRae.,3: 214,1957.
14. Hogan. B. L. M., and Komer, A. Ribosomal Subunits of Landschutz
Ascites Cells during Changes in Polysome Distribution. Biochim. Bio
phys. Acta, 169: 129-138, 1968.
15. Hryniuk, W. M. Purineless Death as a Link between Growth Rate and
Cytotoxicity by Methotrexate. Cancer Res., 32: 1506-151 1, 1972.
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Aced.Sci., 186:330-342,1971.
17. Kalckar, H. M., and Ullrey, D. Two Distinct Types of Enhancement of
GalactoseUptakeinto HamsterCells: Tumor-virusTransformationand
Hexose Starvation. Proc. NatI. Aced. Sd. U. S., 70: 2502-2504, 1973.
18. Kaminskas, E. Serum-mediated Stimulation of Protein Synthesis in
Ehrlich Ascites Tumor Cells. J. BioI. Chem., 247: 5470-5476, 1972.
19. Kaminskas, E. Inactivation of Protein Synthesis Stimulating Activity in
Serumby Cells.J. CellularPhysiol.,82: 475-488,1973.
20. Kaminskas, E. The pH-Dependence of Sugar Transport and of Glycolysis
in Cultured Ehrlich Ascites Tumour Cells. Biochem. J., in press, 1978.
dependent on glycolysis for their energy metabolism. Other 21. Kaminskas,E., Field,M., and Henshaw,E. C. CyclicAMPandGrowthof
tumor cells and normal cells, which depend mainly on
Ehrlich Ascites Tumor Cells. Lack of Cyclic AMP Elevation in Nutrition
respiration for their energy metabolism, may be less sensi
live to depletion of adenylale pools and to inhibition of
glycolysis by MTX. Transformed fibroblasts (34), Novikoff
rat hepatoma cells (29), and reticulocytes (Ref. 7; Fig. 2)
tolerate glucose starvation without decreasing their ade
nylale pools or the adenylale energy charge unless respira
lion is inhibited as well. The mechanism of MTX cytotoxicity
in such cells remains to be investigated; it is possible that it
is due mainly to inhibitions of thymidylate and DNA
syntheses. Therefore, it would be important to ascertain
how common are tumor cells that depend on glycolysis
for their energy metabolism, since these cells would die
when treated with MTX and dThd. The potenliation of MTX
by hypoglycemia and the antagonism of hyperglycemia may
also havetherapeutic implications.
ally DeprivedCellsand Mechanismof Retardationof GrowthbyDibutyryl
CyclicAMP.Biochim.Biophys.Acta,444:539-553,1976.
22. Klein, G. , and Revesz, L. Quantitative Studies on the Multiplication of
Neoplastic Cells in Vivo. I. Growth Curves of the Ehrlich and MCIM
Ascites Tumors. J. NatI. Cancer Inst., 14: 229-277, 1953.
23. Kletzien, R. F., and Perdue, J. F. Induction of Sugar Transport in Chick
EmbryoFibroblastsby HexoseStarvation.J. Biol. Chem.,250:593-600,
1975.
24. Live, T. R., and Kaminskas,E. Changesin AdenylateEnergyChargein
Ehrlich Ascites Tumor Cells Deprived of Serum, Glucose or Amino
Acids. J. Biol. Chem., 250: 1786-1789, 1975.
25. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein
Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265-
275,1951.
26. Martineau, R., Kohlbacher, M. , Shaw, S. N. , and Amos, H. Enhancement
of Hexose Entry into Chick Fibroblasts by Starvation: Differential Effect
of Galactose and Glucose. Proc. NatI. Aced. Sci. U. S., 69: 3407-3411,
1972.
27. Nakamura, W., and Hosoda, S. The Absence of Glucose in Ehrlich
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28. Pinedo, H. N., Zaharko, D. S., Bull, J. M., and Chabner, B. A. The
ACKNOWLEDGMENTS
The authors are indebted to L. Egre for excellent technical assistance.
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CANCER RESEARCH VOL. 38
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
Effects of Methotrexate and of Environmental Factors on
Glycolysis and Metabolic Energy State In Cultured Ehrlich
Ascites Carcinoma Cells
Edvardas Kaminskas and Ann C. Nussey
Cancer Res 1978;38:2989-2996.
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