Rate-limiting Factors in Glycolysis and Transport of Inorganic
Phosphate in DBAH1 Tumor, DBAG Tumor, Novikoff
Hepatoma, and Novikoff Ascites Tumor1
RAY
Department
of Biochemistry,
Wu,
HELEN
The Public
Health
POWER,
Research
AND DAVID
Institute
HAMERMAN2
of The City of New
York,
Inc.,
New
York,
New
York
SUMMARY
In DBAH1 tumor, DBAG tumor, and Novikoff hepatoma, both aerobic and anaero
bic glycolysis were stimulated by increasing the glucose concentration
from 0.5 to 5
mM.
Therefore,
it was
concluded
that
the
availability
of intracellular
glucose
was
a
rate-limiting factor for glycolysis.
In these 3 tumors, the difference between the rates
of aerobic and anaerobic lactate production was primarily due to a more severe inhibi
tion of P-fructokinase3 activity under aerobic conditions.
Thus, P-fructokinase
was at
least partially responsible for the Pasteur effect.
In addition, the inhibition of P-fruc
tokinase resulted in an accumulation of glucose-6-P, which then inhibited hexokinase
activity.
In Novikoff ascites tumor cells, after the addition of glucose, rate-limiting factors for
glycolysis and the levels of intracellular intermediates
were found to change continu
ously with the time of incubation.
Factors responsible for the Pasteur effect also
change with the length of incubation.
Under anaerobic conditions, striking paral
lism between the rate of lactate production and the level of intracellular P, concentra
tion were observed at every time interval.
In all 4 types of tumors, P1 transport can be facilitated by both the glycolytic and
the respiratory energy.
This appears to be a unique feature of all tumors studied so
far, since normal cells examined to date can use only respiratory energy for P1 tramis
port.
Mechanisms
tissues
have
that regulate glycolysis in 4 differemit tumor
been
examined
in order
to identify
the
factors
that control aerobic and anaerobic glycolysis.
The follow
ing factors were analyzed, since one or several of them can
control the over-all rate of glycolysis of the intact cells:
(a) transport
of glucose
into the cells ; (b) availability
investigation
was
supported
by
Public
Health
Service
Research Grant CA-05706 from the National Cancer Institute,
and by Grant DA-AMC-18-035-70(A) from the U.S. Army Chemi
cal Center Procurement Agency.
2 Present
address:
Department
College of Medicine,
3 The
following
of
Medicine,
Albert
Einstein
are
used:
peared
report
on
P
(in
combinations),
phosphate (phospho-); P1, inorganic phosphate; AMP and ATP,
the mono- and triphosphates of adenosine.
Received for publication March 1, 1965.
certain
aspects
of
this
work
has
ap
(19).
MATERIALS
Materials.—Two
types
AND
@IETHODS
of malignant
tumors
were kindly
provided by Dr. A. Goldfeder.
One type is designated
DBAH1 (7), an epithelial slow-growing tumor; the other
type
is designated
DBAG
(7),
a spimidle
cell,
fast-growing
tumor.
Both types were derived from the mammary
gland tissue of the inbred DBA strain of mice and were
transplanted
serially in parent hosts.
The Novikoff hepa
toma (11) and Novikoff ascites tumor cells (3) were kindly
provided by Dr. Villaverdi and were transferred every 5
days into the peritoneal cavity of 150-gm female Sprague
Dawley rats.
Methods.—Slices of DBAH1 and DBAG tumors and of
Novikoff
hepatoma
with a Mcllwain
New York, New York.
abbreviations
liminary
or
transport of P1; (c) availability of cofactors; (d) activity of
glycolytic enzymes ; and (e) presence of enzyme inhibitors
in the cell, especially feedback inhibitors of key enzymes.
In addition to its role in the control of glycolysis, P1 is
important
in many other cellular activities.
Therefore,
Pi transport into these tumor cells was investigated.
Both similarities and differences have been observed in
comparisons of these tumors, with respect to the control
mechanisms
of glycolysis, with one another and with
1 This
Ehrlich ascites tumor cells (21—23),HeLa cells (17), mus
des (10, 14), brain (8), liver amid kidney (20). A pre
Instruments,
Inc.,
were
prepared
mechanical
Great
Neck,
by chopping
tissue chopper
New
York)
the
tissues
(Brinkmann
to a thickness
of 0.416 mm. Conditions for the imicubation of the slices
are given in the tables.
The extracellular
space (irnulin
1733
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1965 American Association for Cancer Research.
TABLE 1
space) was measured with inulin-3H, and this value was
used in the calculation of HP, uptake by the cells of the
GLYCOLYTIC ENZYME PROFILES OF HOMOGENATES OF DBAH1
TUMOR, DBAG TUMOR, N0vIK0FF
HEPATOMA, AND
N0vIK0FF
ASCITES TUMOR CELLS
Enzyme
activites
were measured
at pH 7.4 at 26°C in the for
ward reaction and were expressed as @molesof substrate
min/100
Vol. 25, November 1965
Cancer Research
1734
mg of homogenate
tissue
slices
(18)
and
turnover!
protein.
ascites
tumorDBAGtumorNovoikoff
hepatomaNovikoff
tumor
EnzymeDBAH,
4.0
Hexokinase
36
4.3
66
2.0
Glycolytic
enzyme
72
72
6.6
6.2
33
6.0
5.6
19
Novikoff
hepatoma,
4.1
65
2.7
14
measured
under
88
Hexokinase,
29
35
430
1100
20
29
130
36
32
30
700
56
25
120
140
51
3-phosphoglycerate kinase
3-phosphoglycerate mutase
Enolase
Pyruvate kinase
Lactic dehydrogenase
ATPase
a-Glycero-P
a P,
11
3.3
dehydrogenase
phosphate
(phospho-)
1200
70
44
350
600
130
160
7.3 10
85
; ATPase,
18
1.4
7.0
adenosine
of the
concen
activities
of
glycolytic
of DBAH1 tumor, DBAG tumor,
and
optimal
Novikoff
ascites
conditions,
P-fructokinase,
are
and aldolase
tumor
shown
in
ysis
in these
tumors,
an inhibitor
triphosphatase.
GLYCOLYSIS AND P1 TRANSPORT IN DBAH1
of DBAH1
or DBAG
tumor
(60 mg, wet weight)
unless
their
activity
is depressed
or by a deficiency of cofactor or substrate.
TUMOR AND DBAG
were incubated
TUMOR
at 37°C in 1.8 ml of Krebs
Ringer bicarbonate buffer (P1 = 1.2 mM), pH 7.5. 32@1a
(20,000cpm), glucose-'4C (10 @moles
with 50,000
cpm) and inulin-'H
(0.1 mg with 200,000 cpm) were added to each beaker (20-mi capacity).
were shaken for 40 mm in a Dubnoff shaker.
40 mm.
=
72
of
= 78 mg of protein; for DBAG tumor, 1 gm (wet weight)
protein.
IN COs-N,
IN CO@-O@
C0NcEN
ADDITIONS (os
OMISSION)
TUMOR
TRATION
(mu)
0.2
P1
15
AMP
4
a
70-80%
40
None
IAA
3
per
6
6
5
120
150
6
66
10
of protein
18
6
AMP,
slices
Intracell
ular P@
5
2
8
1
5
12
14
5
10
13
8
8
5
5
75
10
86
9
6
75
72
85
4
1
14
3
76
4
5
8
4
3
53
80
adenosine
in the
144
6
3
6
6
iodoacetate;
amount
uptake
120
155
6
6
40
80
@.smoles
4
65
5
40
as
10
140
Uridine
Imidazole
before
8
115
115
68
protein
140
0
9
13
70
of the
120
6
14
60
IAA,
5
20
7
the
6
5
48
58
represents
Total
Lactate
49
4
phosphate;
Lactate
38
50
57
0.2
Intracell
ular P@
40
15
protein
C Expressed
49
P1
AMP
inorganic
b Final
41
uptake
38
60
5
Uridine
Imidazole
@
lactate
6
7.5
Pr
DBAG
14C
0
IAA
$21,'
Total
Lactate
None
Glucose
omitted
DBAHI
The beakers
Results are expressed as ,@moles/100mg of final protein―!
For DBAH1 tumor, 1 gm (wet weight)
mg
3
monophosphate.
after
the
incubation
and
corresponds
to
incubation.
ml
of
intracellular
1.
were considerably
TABLE 2
Slices
cells,
Table
lower than the other glycolytic enzymes in all 4 tumors.
In DBAH1 and DBAG tumors hexokinase had the lowest
activity; in Novikoff hepatoma and ascites tumor cells
aldolase was the lowest.
Elowever, the hexokinase activ
ity in the homogenates of each tumor was sufficient to sup
port a rate of glycolysis at least 5 times as fast as that ob
served anaerobically in the intact cells. It was therefore
concluded that the enzymes do not limit the rate of glycol
dehydrogenase
isomerase
calculation
profiles.—The
enzymes in homogenates
P-fructokinase
Aldolase
Glyceraldehyde-3-P
in the
RESULTS
Glucose@6@P0i5Omera5e
Glyceraldehyde-3-P
also
trations of intracellular intermediates
and cofactors (19).
Determinations
of total lactate, lactate-14C, and glucose
were the same as previously described (18, 19). Each set
of experiments was repeated at least 3 times.
water.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1965 American Association for Cancer Research.
by
Wii et al.—Rate-limiting Factors in Glycolysis and P, Transport in Tumor Cells
DBAH1 TUMOR AND DBAG TUMOR
only the data for the 40-mm incubation are presented in
Table 2. Glucose utilization by the tumor slices balanced
fairly well with the lactate production, and only data on
the latter are presented.
In slices of DBAH1 tumor, the rate of anaerobic glycol
Control of glycolysis.—It was found that the rate of lac
tate-14C production and P1 uptake were almost propor
tional to time after 20, 40, and 60 mm of incubation, but
ysis
TABLE 3
Slices of DBAH1 or DBAG tumor
(25 mg, wet weight) were
incubated at 37°Cin 1.0 ml of Krebs-Ringer bicarbonate buffer
(in 10-rn! beakers) in a Dubnoff shaker.
After 10 mm of gassing,
varying amounts of glucose-'4C (14,000 cpm/@zmole)were added
through the holes (usually stoppered
with No. 9 rubber stoppers)
on the metal hood for gassing. After the desired incubation
time, 0.5 ml of 20% CuSO4 was added directly to the beakers to
stop the reaction.
The experiments
were planned
so that
at
least 80% of the glucose-14Cremained at the end of the incubation.
Results of averages of 3 sets of triplicate experiments are ex
pressed
as pmoles/100
mg of protein.
PRODUCTIONDBAHI
GLUCOSE-14C
CONCINTRATION
tumorCO2-02C02-N2C02-02C02-N21
TIME (ruin)LACTATE-'4C
(mM)INCUBATION
was
over
tumorDRAG
rate
nucleotides,
these
of aerobic
glycolysis.
of glycolysis
compounds
and
2
8
12.8
21.8
7.3
8
14.0
24.3
4
8
15.0
28.0
7.4
10.3
7.55.3 10.3
5
8
15.8
30.0
10.2
114
60
120
60
5818.4 1184.3
To test
by P@or adenine
other
likely
requirement
for P1 transport.—The
activators
uptake
was studied
with 1.2 nut of P1 in the medium,
amount
of intracellular
HP was measured.
3
30
30
309.6
the
or inhibitors were added to the incubation medium.
Aero
bic glycolysis was found to be stimulated 25 % by the addi
tion of AMP but not by high levels of P1. Unlike the
pronounced inhibition of aerobic glycolysis by uridine or
inosine observed in ascites tumor cells (23) , these nucleo
sides had no effect on the glycolysis of DBAH1 tumor.
In slices of the fast-growing DBAG tumor, the rate of
anaerobic glycolysis was only 25 % higher than the rate of
aerobic glycolysis, as has already been reported by Gold
feder et al. (7). Uridine inhibited both aerobic and anaero
bic glycolysis by 30 %.
In both tumors, imidazole stimulated glycolysis.
The
total lactate values were approximately
20 % higher than
the lactate-'4C values, presumably representing a slow rate
of lactate formed from endogenous substrates.
Energy
5
10
158
twice
for the possible limitation
EFFECT OF GLUCOSE CONCENTRATION ON GLYCOLYSIS OF DBAHI
TUMOR AND DBAG TUMOR
1735
and
Under
bic conditions, P, uptake in both DBAH1 and DBAG tu
mors was found to be low in the absence of glucose or in
the presence of iodoacetate (Table 2). This low level of
uptake occurred in the absence of energy generation and
may be assumed to be due to exchange.
The anaerobic
transport
of P1 was
of glucose.
markedly
Under
aerobic
stimulated
upon
conditions,
the
addition
P1 transport
TABLE 4
CONCENTRATION
Slices
OF
of DBAH1
INTRACELLULAR
or DBAG
tumor
INTERMEDIATES
IN
(45 mg, wet weight)
DBAH1
TUMOR
were inicubated
AND
DBAG
TUMOR
at 37°C in Warburg
vessels.
The final fluid volume was 1.5 ml, which contained 1.3 ml of Krebs-Ringer bicarbonate buffer, pH 7.5;
1.8 .rmoles of @1,a
8 pmoles of glucose-14C (60,000 cpm); and 0.1 mg of inulin-'H (210,000 cpm). Results
represent average values from 3 sets of triplicate experiments.
TUMOR
GAS
LACTATE-―C
PRODUCTION
PHASE
CONTENTS IN
MEDIUM@'
(mismoles)
(p.moles)
CONTENT
%
CO2-02
C02-N2
DBAG
CO2-O2
a P,, inorganic
of
FDP
ATP
FDP
ATP
7.0
7.6
5.0
20
0.17
5.2
20
0.20
0.13
0.14
0.70
0.70
9.6
12
0.06
0.29
0.42
10
0.07
0.36
0.35
0.16
0.14
0.10
1.0
0.034
0.030
0.32
0.6
0.31
0.6
0.6
AMP
and
17
11
12
1.9
17
11
11
20
40
2.1
22
14
10
3.9
4.1
23
13
12
4.8
1.0
1.9
14
14
4.9
31
28
15
10
6.8
22
6.5
6.2
27
19
18
4.2
4.0
13
18
12
17
20
1.6
18
40
3.2
2623
phosphate;
presence
G-6-P
0.9
20
G-6-P,
relatively
large
CONCENTRATION
SAMPLE
20
ATP, the mono- and triphosphates
b The
INTRACELLULAR
40
40
CO2-N2
IN CELLU-
tARSAMPLE
(m@anoles) (pmoles/ml intracellular water)c
IN CELLULAR
G-6-P FDP
DBAH1
12
glucose-6-phosphate;
FDP,
12
fructose
G-6-P
0.11 0.9
1 ,6-diphosphate;
AMP
0.3
of adenosine.
amounts
of
glucose-6-P
and
fructose
1 ,6-diphosphate
in
the
in
cubation medium at the end of the incubation necessitates proper corrections for the determination
and calculation
C Corrected
of the intracellular
concentrations
of P1
the total
anaero
of these glycolytic
intermediates
(19).
values.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1965 American Association for Cancer Research.
was
Cancer Research
1736
quite rapid amid was further increased upon the addition
of glucose.
In the presence of both glucose and iodoace
tate,
aerobic
P1 uptake
was
even
greater.
This
and
in intracellular
levels
of P1 were
increased
observed
1 , 6-di-P
, 6-di-P,
and
ATP
were
similar
after
when
20 and
level
was
3 times
as
high,
indicating
that
P-fructo
kinase activity was lower under aerobic conditions.
A
higher level of ATP and a lower level of AMP (13) under
aerobic conditions were also consistent with the interpre
tation that in both DBAH1 and DBAG tumors, aerobic
glycolysis was controlled, at least in part, by the inhibition
of P-fructokinase
activity (19). In the presence of a low.
level of aerobic AMP, the inhibition of this enzyme by
high ATP may be partly responsible for the Pasteur effect.
higher concentrations
of P1 were employed in the medium.
Effect of glucose concentration on glycolysis.—In
i
tumor (Table 3), both the aerobic and anaerobic lactate
production increased by about 50 % when the glucose con
centration was increased from 1 mi@ito 5 m@m. Therefore,
below 5 mM, the availability of glucose was a rate-limiting
factor.
In the DBAG tumor glycolysis was already max
imal at 2 mM glucose.
Control of aerobic glycolysis by P-fructokinase.—The
levels of intracellular
intermediates
under aerobic and
anaerobic conditions were then analyzed in attempts to
locate the rate-limiting
step in aerobic glycolysis.
As
shown in Table 4, the intracellular levels of glucose-6-P,
fructose-i
N0vIK0FF
HEPATOMA
AND NOvIKOFF
ASCITES TUMOR CELLS
Control of glycolysis.—The
slices
of Novikoff
rate of anaerobic
hepatoma
(Table
glycolysis
5, Experiment
40
Pi
were
appreciably
increased.
After
gentle
homogeniza
tion of Novikoff hepatoma (Table 5, Experiment 2), the
isolated tumor cells showed an increased aerobic glycolysis
TABLE 5
GLYCOLYSmS AND P, TRANSPORT IN NOvIKOFF HEPATOMA AND NOVIKOFF
CELLS
For Experiment
1, slices
For Experiment
down strokes,
the mixture
of Novikoff
hepatoma
(70 mg, wet weight)
2, the slices were homogenized
was diluted
10 times
ASCITES TUMOR
were incubated
as described
in
in a loose glass homogernizer with 3 up and
with Krebs-Ringer
bicarbonate
buffer
and centrifuged,
and the packed cells (at least 50% viable cells) were resuspended and used. For Experiment 3, Novikoff
ascites tumor (5 mg of cell protein/experiment) was used. Results are expressed as Mmoles/ml of packed
cells/40
ascites
mm.
tumor
For Novikoff
hepatoma
slices, 1 gm (wet weight)
cells, 1 ml of packed cells = 120 mg of protein.
= 95 mg of protein;
IN CO2-O2
EXPERI
MENT
(mu)
None
Glucose
omitted
IAA
0.1
Pa
P.
AMP
2
Total
32p1a
Intracell-
lactate uptake
ular p@b
Lactate-
Total
% PAS.
@C lactate
“P@ Intracell
uptake
ular P@
1.6
0.4
8.5
8.5
7.5
40
43
0
2.6
1.5
6.3
9.0
98
106
4
0
0
4.0
2.5
0
0
0.3
EFFECT―
60
10
42
46
5.6
7.8
102
110
5.0
10.2
59
20
40
46
10.0
9.8
98
108
7.5
11.3
4
47
60
53
None
Pr
AMP
3
IN CO@-N@
TEUR
Lactate-
14C
1
for Novikoff
CONCEN
TRATION
ADDITIONS
(oR
OMISSIONS)
20
4
None
2.3
101
1.4
68
70
3.4
116
124
2.3
42
64
64
68
9.5
3.0
112
100
120
9.0
1.8
43
116
124
0
6.7
5.2
14
13
225
225
9
0
5.4
13
Glucose
36
6.3
3.0
15
24@
2.8
22
49
omitted
IAA (glucose
0.3
omitted)
P@
P1
a p,@ inorganic
b Expressed
C Percent
anaerobic
Pasteur
12
122
134 17.5
23
225
228 15
21
124
132 23
30
222
227 22
32
IAA,
pmoles/ml
effect
4
24
phosphate;
as
of
on
iodoacetate;
intracellular
lactate-'4C
AMP,
adenosine
46
44
monophosphate.
water.
production
in
1) was
2.5 times as high as that of aerobic glycolysis.
Lactate
production was not stimulated by AMP or high levels of
Pi, even though in the latter case the intracellular levels of
mm of incubation, indicating that steady-state
levels had
been reached in 20 mm. In spite of the higher rate of
Table 2.
1965
glucose phosphorylation
under anaerobic conditions, the
intracellular
level of glucose-6-P was found to be much
lower anaerobically than aerobically, whereas the fructose
uptake was presumably due to the accumulation of large
amounts of fructose-i ,6-di-P (22).
Large increases in both HP, uptake (transport amid diffu
sion)
Vol. 25, November
=
100
X
(anaerobic
lactate
—
aerobic
lactate)/
lactate.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1965 American Association for Cancer Research.
Wij et al.—Rate-limiting
Factors in Glycolysis and P, Transport
in Tumor Cells
1737
TABLE 6
EFFECTS
Slices
of Novikoff
as pmoles/ml
intracellular
OF INHIBITORS
hepatoma
AND ACTIVATORS
were incubated
of packed cells/40 mm, arid intracellular
ADDITIONSIN
HEPATOMA
5. Lactate
@1a
concenitration
production
is expressed
is expressed
as @.amoles/mlof
C02-O2IN
(mM)Lactate
14CTotal
concentrationNone
Inosine
Pr
33
5.4
98
108
8.0
20
32
4.0
90
1038.0
7.0
11
30
34
13.2
99
22
325.2
13.0
96
5.498
100
5 + 11
50
0.15
Dinitrophenol5
inorganic
48
105
0.2030
8.1
90
7534
80106
phosphate.
TABLE
EFFECT
lactateIntracellular @1
26
Uridine+ Pj
Imidazole
Dinitrophenol
14CTotal
lactateIntracellular
concentrationLactate
5
Uridine
Slices
ON N0YIKOFF
in Table
water.
CO2-N2CONCENTRATION
a P1@
as described
7
OF GLUCOSE CONCENTRATION ON GLYCOLYSIS IN N0YIK0FF
NOVIKOFF ASCITES TUMOR
of Novikoff
hepatoma
(50 mg, wet weight)
or Novikoff
ascites
HEPATOMA AND
tumor
cells
(3 mg of proteini)
were incubated at 37°Cin 2.0 ml of Krebs-Ringer bicarbonate buffer (in 20-mi beakers) in a Dubnoff
shaker. Other conditions were the same as described in Table 3, except that the incubation time was
10 mm. With Novikoff hepatoma, identical experiments were run, but the hepatoma was deprotein
ized with HC1O4; after neutralization
the supernatent solution was used for pyruvate determination
(with reduced diphosphopyridine
@tmo1es/100mg of protein/lO
nucleotide and lactic dehydrogenase).
LACTATE-―C
GLUCOSE-14C
CONCENTRATION
TUMOR
PRODUCTION
% PASTEUR
CO2-O2
Novikoff hepatoma
ascites
0.5
1
2
3
4
6
CO,-N@
Pi
levels,
did
not
glycolysis
appreciably
(40-mm
affect
the
incubation).
14
63
5.7
6.1
21
6.8
8.4
24
28
28
71
71
6.7
7.4
70
70
9.0
9.0
8.5
47
46
48
47
25
24
25
25
over-all
As
rate
of
will
be
showed later (Table 12), P1 was limiting between 1 and 8
xthn of incubation.
The values of the total lactate were
only slightly higher than the lactate-'4C, indicating little
or no glycogenolysis(1).
PYRUVATE
PRODUCTION
CO2-N2
CO,-O2
C02-N2
2.8
3.0
0.16
0.22
0.8
0.10
4.3
5.2
and the Pasteur effect was lowered from 60 % to 40 %.
Glycolysis in Novikoff ascites tumor cells (Table 5, Ex
periment 3) was more than twice as high as in Novikoff
hepatoma (hexokinase activity in Novikoff ascites tumor
cells was 8 times as high), and the % Pasteur effect was
slightly lower in the ascites tumor cells. High levels of P1
in the medium, which greatly increased the intracellular
long-term
CO202
4.0
1
2
4
6
tumor
TOTAL LACTATE
PROCUCTION
EFFECT
(mM)
Novikoff
Results are expressed as
mm.
47
48
48
47
15
21
26
28
29
26
26
50
50
26
49
26
48
Aerobic glycolysis, as measured by lactate-'4C produc
tion, was slightly inhibited by inosine and appreciably in
hibited by uridine (Table 6). Inhibition by uridine was
observed even in the presence of 11 m@iP1 in the medium.
In contrast, in Ehrlich ascites tumor cells, inhibition of
glycolysis by uridine was reversed by 11 m@ P1 (23).
Total lactate production in Novikoff hepatoma was not
inhibited by inosine or uridine, owing presumably to the
formation of lactate from the pentose moiety of the added
nucleosides.
Both
imidazole
lated aerobic glycolysis,
anaerobic glycolysis.
Energy
requirement
and
dinitrophenol
but the latter
slightly
for P transport.—The
stimu
inhibited
P1 transport
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1965 American Association for Cancer Research.
in
TABLE
CONCENTRATION
OF NOVIKOFF
INTERMEDIATES
IN
HEPATOMA
of Novikoff
hepatoma
(90 mg, wet weight)
at 37°C inn 1.5 ml of Krebs-Ringer
bicarbonate
1965
In Novikoff ascites tumor cells, in contrast to the solid
tumor, glycolysis was independent of the glucose concen
tration between 1 and 6 m@.
8
OF INTRACELLULAR
SLICES
Slices
cubated
@
Vol. 25, November
Cancer Research
1738
were in
buffer.
Control of aerobic glycolysis
fructokinase.—The
intracellular
of Novikoff hepatorna by P
levels of sugar phosphates
After 10 mm of gassirng, 9 @moles
of glucose-―C(8000cpm/@rmole) and adenine nucleotides in Novikoff hepatoma (Table 8)
and 0.1 mg of inu!in-'H
(210,000 cpm) were tipped in from the
were similar after 10, 25, and 40 mm of incubation.
Un
double side arm of the Warburg
vessel.
At the end of incubation,
der
aerobic
conditions,
the
level
of
fructose-6-P
(substrate
the medium was tipped into the double side arm before 0.1 ml of
for P-fructokinase)
was found to be higher than under
10 N HC1O4 was tipped from the single side arm irnto the main
anaerobic conditions ; in contrast, the level of fructose
compartment
to deproteinize
the tumor slices (19). Results
are
1 , 6-di-P (product of P-fructokinase
reaction) was lower.
expressed as ,@moles/gm of slices (wet weight), and only the cor
Therefore, under aerobic conditions, as in DBAH1 and
rected intracellular concentrations of intermediates are presented
here (see Table 4 and Ref. 19).
DBAG
tumors,
P-fructokinase
appeared
to be
a rate-lim
iting factor of glycolysis in Novikoff hepatoma.
A higher
CONCENTRATION
aerobic level of ATP and a lower level of AMP also favor
values)Glucose
(corrected
Ii@ PRO
GASPHASEINCUBATION
the inhibition of P-fructokinase
(13, 19).
UPTAKEINTRACELLULAR
TIME (mm)LACTATE
DUCTIONGLUCOSE
Effect of glucose concentration on the intracellular inter
@@paFructoseFDPATPAMPCO2-O210160.240.080.081.60.0725
mediates in slices of Novikoff hepatoma.—The experiments
shown in Table 8 were carried out in the presence of high
levels of glucose.
The effect of lowering the glucose con
centration
on the rate-limiting
factor of glycolysis in
1.70.07C02-N210480.090.030.220.70.2425
0.101.6
66460.27
0.230.09 0.090.08
4034
Novikoff hepatoma is illustrated in Table 9. Upon in
creasing the levels of glucose up to 6 mr@i,the levels of in
tracellular hexose-mono-P
and -di-P are both increased
0.210.7
0.60.25 Therefore, when glucose is limiting, increasing the levels
0.090.03
0.030.20
4098
1561100.10
of glucose makes the rate of glycolysis directly propor
a
(in combinations),
phosphate;
FDP,
fructose
1,6-diphos
tional to the level of glucose-6-P, either aerobically or
phate; AMP arid ATP, the mono- and triphosphates of adenosine.
anaerobically.
However, at any given level of glucose, the
aerobic
hexose-mono-P
level is always higher than that
TABLE 9
under
anoxia
;
thus
the
rate
of glycolysis is inversely pro
EFFECT
OF GLUCOSE
CONCENTRATnON
ON THE
INTRACELLULAR
portional to the levels of glucose-6-P when aerobic and
INTERMEDIATES IN SLICES OF NovnKoFF HEPATOMA
Slices
of Novikoff
Warburg
hepatoma
vessels as described
time was 10 mm.
(80 mg) were incubated
at 37°C in
TABLE
in Table 8, except that incubation
KINETICS
Results are expressed as in Table 8.
OF GLYCOLYSIS
INTERMEDIATES
AND
IN
10
ACCUMULATION
SLICES
OF
OF INTRACELLULAR
N0vIK0FF
HEPATOMA
Slices of Novikoff hepatoma (75 mg) were used. Experimental
values)COt-O,CO2-N2CO202CO2-N@HMP―FDPbHMP―FDP@18.3210.340.0180.0750.08829.6340.400.0270.0920.133310.6380.460.0380.1140.140411.7430.500.0530.1400.154611.7430.510.0600.1500.150
CONCENTRATION(corrected
GLUCOSE
conditions
PRODUCTIONINTRACELLULAR
CONCEN
TRATION
(mM)LACTATE-14C
in Table
were the same, and results are expressed,
as described
8.
CONCENTRATIONTotalPer
GAS PHASEINCUBATION
TIME (mm)LACTATE-'4CINTRACELLULAR
mono
mm―Hexose
phosphatesFructose
diphosphateC02-O20
1,6-
1
1.7
a HMP,
hexose
monophosphates,
consistirng
of
glucose
6-phos
4
phate (75%) and fructose 6-phosphate (25%).
b FDP,
fructose
5)
was
similar
to
10
amidNovikoff ascites tumor cells (Table
that
in
DBAH1
and
DBAG
0.50
0.068
0.51
0.066
1.0
1,6-diphosphate.
Novikoff hepatoma
8.3
tumors
14.2
1.080.50
0.065C02-N20
203.2
25.03.2
0.520.044
in
that the 32p1 transport was supported by both glycolysis
and respiration.
Effect of glucose concentration on glycolysis.—Both the
aerobic and the amiaerobic glycolysis of Novikoff hepatoma
increased appreciably with increasing glucose concentra
tion from 0.5 to 4 mM (Table 7). Under aerobic condi
tions, large amounts of pyruvate (amounting to about 30 %
of lactate production)
were formed, indicating that the
generation of DPNH may be insufficient.
Only a very
small amount of pyruvate was formed anaerobically.
1
2.8
4
13.4
0.11
0.13
0.13
0.14
0.140.11
0.14
3.1
10
32.0
3.00.06
205.0
a Micromoles/min
62.05.0
between
2
time
intervals.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1965 American Association for Cancer Research.
Wu et al.—Rate-limiting
TABLE
KINETICS
OF GLYCOLYSIS
AND
Factors in Glycolysis and P, Transport in Tumor Cells
11
of glucose ; in anoxia, the steady
ACCUMULATION
OF INTRACELLULAR
1739
state was reached
after
iO mm.
INTERMEDIATES
IN N0vIK0FF
ASCITES
TUMOR
CELLS
In Novikoff ascites tumor cells (Table 11), the fructose
Novikoff ascites tumor cells (7 mg of cell protein) were in
1 , 6-di-P
level
became
maximum
between
2 and 10 mm but
cubated in 2 ml of Krebs-Ringer bicarbonate buffer at 37°C. fell abruptly and continuously between 10 and 40 mm of
After 10 mm of gassing, glucose (10 @moles)was tipped in from incubation.
The level of triose-P paralleled the levels of
the double side arm of the Warburg vessel. To terminate the
fructose-i , 6-di-P, and the equilibrium constant calculated
incubation at various time intervals, 0.08 ml of 10 N HC1O4was
for aldolase reaction remained steady during the entire
tipped into the main compartment
from the single side arm.
period of incubation.
As in Novikoff hepatoma, at any
Results are expressed as pmoles/ml of packed cells.
given time the aerobic hexose-mono-P
level was always
higher
than
in
anoxia,
whereas
the
fructose-i
, 6-di-P level
CONCENTRATIONGAS
PRODUCTIONINTRACELLULAR
was
lower.
It
was
noted
that
the
intracellular
level
of
TIME(mm)TotalHMP@'FDPTriose
PHASEINCUBATION
fructose-i , 6-di-P was extremely high when the rate of
j@hos@
glycolysis was lowest.
This and other aspects of the un
atesATPCO2-O205.515.52.92.81.41.62.027.52.92.91.41.81.149.71.83.01.52.33.710321.63.01.42.83.820701.71.40.93.03.6301061.80.80.63.13.4401401.90.70.53.0C02-N,0141140.64.11.70.662200.66.12.40.54.54290.66.02.4
p
usually large fluctuation of the levels of intracellular corn
pounds in Novikoff ascites tumor cells were then analyzed
in greater
detail.
Control of aerobic glycolysis
in Novikoff
ascites tumor cells.
—As shown in Table 12, the rates of both glucose uptake
and lactate production were slowest between 1 and 2 mm
after glucose addition.
The slow rate of glucose uptake
may be correlated with the high intracellular levels of glu
cose-6-P, which inhibited hexokinase, and with the rela
tively low level of P,, which was insufficient to reverse this
inhibition (15). The slow rate of lactate production may
be correlated with the low levels of intracellular P1, which
partly
limits
glyceraldehyde-3-P
dehydrogenase
(P@
dropped to half the initial value after 1 mm). it must be
emphasized that other factors may also play a role in regu
lating the activity of this enzyme.
For example, it has been
suggested that an inhibitor of this enzyme is produced
during mitochondrial
respiration (2). It is clear that P
fructokinase must be limited to the same extent, since there
was
no
increase
in
the
level
of
fructose-i
, 6-di-P.
The
higher rate of glycolysis as observed after 6 mimi may be
corrected with a higher level of P1 and lower levels of
hexose mono-P and -di-P. A slight increase in the activ
ity
of glyceraldehyde-3-P
dehydrogenase
an 80 % increase in the glycerate-3-P
as compared
to 4 mm.
This
increase
was
indicated
by
level found at 6 miii
occurred
at
a time
when the rate of conversion of glycerate-3-P to lactate was
also increased, clearly indicating that the glyceraldehyde
3-P
a Micromoles/min
I, HMP,
phate;
Hexose
between
ATP, adenosine
C Triose
phosphates
dihydroxyacetone
2 time
intervals.
monophosphates;
include
anaerobic
experiments
, 6-di-P
Fructose
1 ,6-diphos
triphosphate.
phosphate
fructose-i
FDP,
glyceraldehyde
3-phosphate
in approximately
are compared.
is always
lower
under
and
1: 10 ratio.
Since the level of
aerobic
conditions,
it indicates that the P-fructokinase
step is a rate-limiting
step for aerobic glycolysis at both low and high glucose
concentrations.
Kinetics
of the accumulation
of glycolytic
dehydrogenase
step
was
accelerated.
the activity of this enzyme must
P-fructokinase
to account for the
tose-i , 6-di-P level. These data
the rate-limiting factor for aerobic
intermediates.—
The extent of accumulation of intracellular intermediates
in slices of Novikoff hepatoma was measured between 1 and
20 mm. Data in Table 10 indicated that at optimal levels
of glucose, under aerobic conditions, both hexose-mono-P
and -di-P reached a constant level 4 mm after the addition
1 and
4
mm
was
hexokinase
The
increase
in
be greater than that of
slight drop in the fruc
therefore suggest that
glucose uptake between
and
P-fructokinase,
while
limitation for lactate production was due mainly to glycer
aldehyde-3-P dehydrogenase.
However, a change of lim
iting factors occurred later on.
Control of anaerobic glycolysis in Novikoff ascites tumor
cells.—Both the rate of glucose uptake and that of lactate
production were slowest between 3 and 8 mm, which may
be correlated with the precipitous drop in the intracellular
concentration of P1 and the accumulation of large amounts
of fructose-i , 6-di-P.
The explanation
of fructose-i , 6di-P accumulation must be that it is utilized at a slower
rate than it is formed.
The slow rate of its utilization
appeared
to
hyde-3-P
dehydrogenase
be
the
result
of an
inhibition
by a lack of P,.
of glyceralde
As a conse
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1965 American Association for Cancer Research.
Cancer Research
1740
TABLE
RATE-LIMITING
Novikoff
ascites
FACTORS
tumor
bonate buffer and treated
as
indicated.
An
identical
FOR
cells
AEROBIC
set
of
ANAEROBIC
TUMOR
CELLS
GLYCOLYSIS
were
incubated
in Table 11, except that
Warburg
vessels
1965
12
AND
(16 mg of cell protein)
as described
Vol. 25, November
was
used
for
IN
ASCITES
in 2 ml of Krebs-Ringer
the amount
the
N0vIK0FF
bicar
of added glucose varied
determination
of
inorganic
phos
phate (P1) in the medium; at the end of incubation, the tumor cells were poured into chilled centrifuge
tubes and centrifuged
immediately
for 2 mm; the supernatant
solution was deproteinized
roacetic acid and used for the determination of P1 of the incubation medium.
with trichlo
Results are expressed
as @imoles/ml of packed cells.
GAS PHASE
BATION
TIME
(mm)
CO2-O2
LACTATE
PRODUCTION
GLUCOSE
UPTAKE
INCU
0
GLUCOSE
ADDED
(mM)
Per
Total
n@1ra
5.2
8.0
2.0
1.6
3
2.0
10.8
4
2.0
13.4
12
3.0
34
2.7
56
0
0
2.5
20
2.5
24
4
3.0
27
38
12
4.0
53
mini
between
b Represents
P,
2
time
correct for the intracellular
C HMP,
hexose
monophosphates;
1.44
3.3
2.3
0.05 2.5
6
1.40
3.1
2.2
0.05 3.0
7
2.5
2.3
0.05 3.2
8
4.4
8
12
2.1
2.2
0.05
9
4.5
17
25
1.7
1.9 0.09 3.6 11
1.25
1.7
1.7
4.7
29
37
4.0
31
50
1.10
1.7
1.5 0.09 3.8 14
1.05
1.5
1.0 0.08 3.8 15
1.52
0
0
0
1.44
0.50
2.2
0.14 0.54 14
1.41
0.60
5.6 0.14 1.4
8
0.60
8.2
3
0.62
9.0 0.07 1.8
3
0.60
8.6
0.07
4
1.28
0.60
7.6
0.07 2.3
5
1.23
0.66
4.8
0.07
2.5
8
1.23
0.80
3.9
0.08 2.8
9
1.40
44
3.4
0.09 3.6
11
0.1020
0.10
1.6
2.2
6
56
7.5
86
8.0
150
84
concentration
0
5
3.9
a Per
0
4
3.8
6.0
10
34
3.0
4.0
4.4 13
0
30
32
8
P1
6
2.5
20
13
24
3
3.5
ATP
12
4
6
3.6
12
3
3-PGA
12
11
2.5
44
0
10
2
60
84
11
1
3.4
52
2.7
0
75
33
23
FDP
1.47
24.0
18
2.5
HMPC
1.30
2.5
CO2-N,
88
15.2
2.3
8
(mu)
11.6
2.6
5.0
3.0
8.2
9.2
2.5
CONCENTRATION
5.2
1.2
20
Lactate
tion
pr@uc
0
1.5
6
Glucose
min'@ uptake
8.0
2
INTRACELLULAR
MEDIUM@@
Total
0
1
PASTEUR
EFFECT― %
intervals.
in
the
medium
at
the
ernd
of
incubatiorn.
These
values
were
used
to
P1 contents.
FDP,
fructose
1,6-diphosphate,
which
included
triose
phosphates;
3-PGA, 3-phosphoglyceric acid; ATP, adenosine triphosphate.
quence, the accumulation
of fructose-i ,6-di-P further
lowered the intracellular P@level. The P1 level at 3 mm
wa.s so low that inhibition of glyceraldehyde-3-P
dehydro
genase may be sufficient to accoumit for the inhibition of
lactate production.
This interpretation
is supported by
the fact that the rate of lactate production closely par
alleled the level of P at every time interval.
Since P, is
found to partially reverse the glucose-6-P inhibition of
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1965 American Association for Cancer Research.
Wu et al.—Rate-limiting Factors in Glycolysis and P, Transport
Novikoff ascites tumor hexokinase (R. Wu : to be pub
lished) the low level of P; would allow glucose-6-P to
inhibit hexokinase more effectively to account for the
severe inhibition of glucose uptake.
As the ATP level
became sufficiently high (at about 6 mm) to inhibit P
fructokinase
activity appreciably,
the fructose-i ,6-di-P
level began to fall, and the release of its phosphate gradu
ally increased the P1 level. It is likely that the increase in
Pi would accelerate glyceraldehyde-3-P
dehydrogenase
activity and lactate production,
on the one hand, and
partly overcome the glucose-6-P inhibition of hexokinase
and increase glucose uptake, on the other.
Therefore,
the major rate-limiting factor for anaerobic glycolysis was
gradually shifted from glyceraldehyde-3-P
dehydrogenase
(ist 6 mm) to P-fructokinase
(after 6 mm).
The Pasteur effect in Novikoff ascites tumor cells.—Data in
Table i2 show that the % Pasteur effect on glucose uptake
and lactate production
varies continuously
with time;
therefore, in considering the factors responsible for the
Pasteur effect one must specify the time period under comi
sideration.
The interpretation
of the results is based on
the findings that inhibition of P-fructokinase
activity by
can be relieved by AMP, P1, or fructose-i ,6-di-P (i3) and
that inhibition of hexokinase activity by glucose-6-P can
be relievedby P1 (15).
The following events appear to be pertinent in explain
ing why the Pasteur effect is high between i and 3 mm
(a) The
level
of aerobic
that under
anaerobic
sistent
the
with
fact
glucose-6-P
conditions.
that
hexokinase
is much
higher
This difference
activity
than
is con
is less
inhib
ited and glucose uptake is faster anaerobically than aero
bically.
(b) The lower anaerobic glucose-6-P level under
conditiomis of a faster rate of glucose phosphorylation
can
be attributed only to a faster rate of hexose-P utilization,
which is catalyzed by glucose-6-P isomerase and P-fructo
kinase.
This explanatiomi is supported by the fact that the
level of fructose-i ,6-di-P is higher anaerobically.
(c) The
isomerase is not likely to be involved, since the concentra
tions of glucose-6-P and fructose-6-P are close to equilib
rium.
Therefore, P-fructokinase is involved in the control
of the rate of hexose-P phosphorylation.
(d) The rela
tively higher activity of P-fructokinase under anaerobic vs.
aerobic conditions may be explained by a more favorable
balance of activation over inhibition of this enzyme under
anaerobic conditions.
Thus, the relatively lower anaero
bic levels of ATP, together with higher levels of Ai\1P, may
be responsible for the higher activity of this enzyme.
The
relatively lower anaerobic levels of fructose-6-P and P1 are
apparently not so important in controlling the enzyme ac
tivity.
(e) Since the rate of lactate formation is faster
anaerobically
than aerobically, all the enzyme steps be
tween aldolase and lactate dehydrogenase
must be faster
anaerobically.
The % Pasteur effect is lower (below 20 %) between 3
and 8 mm. This may be explained as follows: (a) Al
though the level of glucose-6-P is lower anaerobically, P1
is also
much
lower.
The
net
effect
of these
compounds
on
hexokinase is to inhibit the enzyme nearly as strongly an
aerobically as aerobically to account for the similar rates
of glucose phosphorylation.
(b) Within this time interval,
the level of fructose-i ,6-di-P remained essentially con
stant,
both
aerobically
1741
in Tumor Cells
and
anaerobically,
amid started
to
decrease only after 6 mm. Although the anaerobic level
of fructose-i ,6-di-@ is higher, P, is lower. Therefore,
there is no indication that P-fructokinase
activity is much
higher anaerobically.
(c) The level of substrate
for
glyceraldehyde-3-P
dehydrogenase is higher anaerobically,
but the level of P1 is much lower. As a result, the activity
of this enzyme is only slightly higher anaerobically.
In
summary, during this time interval, P, had a cont@oIlimig
influence on 3 key enzymes—namely, hexokinase, P-fructo
kinase, and glyceraldehyde-3-P
dehydrogenase.
The % Pasteur effect increased to approximately
40 %
between 8 and 20 mm. (a) The difference between the
aerobic and anaerobic levels of glucose-6-P is not nearly as
great as between 1 and 3 mm, but the lvels of P, and ATP
are lower anaerobically.
As a result, the rate of anaerobic
glucose phosphorylation
is only 30 % higher than under
aerobic conditions.
(b) Other aspects of the Pasteur effect
are quite similar to those between 1 and 3 mm.
DISCUSSION
The analysis of the rate-limiting factors of glycolysis in
the DBAH1 and DBAG tumors, Novikoff hepatoma, and
Novikoff ascites tumor cells revealed remarkable similari
ties
among
that
the
DBAH1
3 types
tumor
adenocarcinoma,
of solid
tumors,
is a relatively
that
in spite
of the
slow-growing
DBAG
tumor
is
a
facts
mammary
fast-growing
spindle cell type of mammary sarcoma, and that Novikoff
hepatoma is originally a rat liver tumor induced by azo
dye. Although Novikoff ascites tumor was derived from
the hepatoma, the control of glycolysis in these 2 tumors is
quite different.
In all 3 solid tumors, both aerobic amid anaerobic glycol
ysis
are stimulated
in the medium,
these tumors.
DBAG
tumor
by increasing
the
glucose
concentratiomi
although
the saturation
level differs in
It is interesting
that
the fast-growimig
reaches
maximal
rate
of
glycolysis
at
a
lower level of glucose (2 mM), whereas the slower-growimig
DBAH1
tumor
requires
5
nmm of
glucose
for
(the level of blood glucose is approximately
saturation
5 mM).
These
results
a high
imidicate that either
the intracellular
hexokimiase
has
Km value
or glucose
transport
is rate limitimig,
or
both.
For hexokimiase in the homogenate
hepatoma,
maximal
the Km value
for
velocity
of hexokinase
glucose
in the
attaimied below i m@i of glucose.
tissue,
glucose
20).
however,
may
have
than
does hexokinase
Nevertheless,
it
may
The hexokinase
a much
inn the
be
of Novikoff
was 0.09 mM ; and
cell-free
system
was
in the
higher
Km value
homogenates
(9,
concluded
that
the
for
12,
intra
cellular glucose was too low to saturate the intracellular
hexokimiase.
In all 3 solid tumors, at saturation
levels of glucose,
aerobic glycolysis appeared to be comitrolled mainly by
P-fructokinase.
increased
glucose-6-P
instead
The
the
level
inhibition
of this
of glucose-6-P.
reached
of increasing
a steady-state
indefinitely,
also
have
of P-fructokinase
been
inhibited.
the
by
ATP
imitracellular
level in 20 mm or less
and
in these tumors was also inhibited
must
enzyme
Since
since
glucose
aerobically,
Thus
a primary
under aerobic conditions
uptake
hexokinase
inhibitiomi
resulted
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1965 American Association for Cancer Research.
in an
1742
increase of the glucose-6-P level, which then led to the well
known product inhibition of hexokinase (4, 16).
At
Vol. 25, November 1965
Cancer Research
suboptimal
levels
of glucose,
aerobic
glycolysis
was
also controlled by P-fructokinase ; an inhibition of this
enzyme also increased the level of intracellular glucose-6-P
and resulted in an inhibition of hexokinse.
Therefore,
hexokinase was actually restricted by both the suboptimal
intracellular level of glucose and the product inhibition of
glucose-6-P.
In considering the factors responsible for the Pasteur
effect, one is really interested in comparing the limiting
step umider aerobic conditions with that under anaerobic
conditions.
Since this comparison is on a relative basis,
the factor responsible for the Pasteur effect may not nec
essarily be the only existing rate-limiting factor for either
aerobic or anaerobic glycolysis.
For example, glucose
transport appears to be a major rate-limiting
factor for
aerobic as well as anaerobic glycolysis in Novikoff hepa
toma (Table 7). Glucose transport, however, is not re
sponsible for the Pasteur effect, as the % Pasteur effect
remained constant with low and high levels of glucose.
In studying the short-term kinetics of glycolysis, one
may find changes in the rate-limiting factors.
The limit
ing factor during the 1st few mm may not bear any relation
to the limiting factors that control glycolysis under steady
state conditions (Table i2). It is obvious that short-term
aerobic studies alone do not provide sufficient information
for speculation on what factor may be responsible for the
Pasteur effect in short-term or long-term experiments.
Pm transport
was
facilitated
by
either
glycolysis
or res
piration in all 3 solid tumors and in Novikoff ascites tumor
cells, similar to the results with Ehrlich ascites tumor cells
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tumor growth.
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of the Aerobic Inhibition of Glycolytic
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prepared
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negligible oxidative phosphorylation
in the absence of
added serum albumin.
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are able to use respiratory energy for P1 transport ; there
fore, oxidative phosphorylation
in these tissues is func
tionally coupled, and damage must have occurred during
the homogenization of the tissue and the isolation of mito
chondria from these tumors.
This conclusion is consistent
with the observations of Goldfeder and Miller (6), indicat
ing that the mitochondria
of the DBAG tumor are more
fragile as compared to those of DBAH1 tumor.
ACKNOWLEDGMENTS
The authors wish to extend their thanks and appreciation to
Dr. Anna Goldfeder, who provided them with DBAH1 and DBAG
tumors; and to Dr. Villaverdi for the strains of Novikoff hepatoma
and Novikoff ascites tumor cells.
15: 33—37, 1964.
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H.,
and
Bone,
A. D. The
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pp. 265-88. New York: Ronald
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1965 American Association for Cancer Research.
Rate-limiting Factors in Glycolysis and Transport of Inorganic
Phosphate in DBAH 1 Tumor, DBAG Tumor, Novikoff Hepatoma,
and Novikoff Ascites Tumor
Ray Wu, Helen Power and David Hamerman
Cancer Res 1965;25:1733-1742.
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