Download Growth-related Enzymatic Control of Glycogen

[CANCER RESEARCH 44, 154-160, January 1984]
Growth-related Enzymatic Control of Glycogen Metabolism in Cultured
Human Tumor Cells1
Monique Roussel,2 HervéParis, Guillemette Chevalier, Blandine Terrain, Jean-Claude Murat, and
Alain Zweibaum
Unitéde Recherches sur le Métabolismeet la Différenciation de Cellules en Culture, INSERM U178, Hôpital Broussais, 96 rue Didot, 75674 Paris Cedex 14 ¡M.ft, G. C
A. Z.], and UniversitéPaul Sabatier, Institut de Physiologie, 2 rue FrançoisMagendie, F-31400 Toulouse [H. P., B. T., J-C. M.], France
ABSTRACT
The activities of glycogen synthase and phosphorylase were
measured and compared to the growth-related variations of
glycogen accumulation in three cultured human tumor cell lines:
HT-29 (colon carcinoma); MeWo (malignant melanoma); and RT4 (carcinoma of the urinary bladder). A similar pattern of varia
tions in the enzyme activities was found in the three cell lines.
The activities of the a + b forms of glycogen phosphorylase
increased throughout the culture period. Maximal activity of
phosphorylase a coincided with low intracellular concentrations
of glycogen during the period of exponential growth. When the
rate of cell division decreased, phosphorylase a activity also
decreased while the glycogen levels increased. Glycogen syn
thase was almost entirely in b form during the entire culture
period, i.e., in both the exponential and the stationary phases. In
vitro incubation of the cellular extracts without NaF showed,
however, that the enzyme could be partially converted to the a
form by the endogenous phosphatases. The Ao.5values of the
enzyme for glucose-6-phosphate (Glc-6-P) were of the same
order of magnitude as the intracellular Glc-6-P concentrations
which ranged from 2.2 to 5.4 mw (almost 10 times those reported
in normal cells). Similar Glc-6-P values were obtained by two
different extraction methods controlled by the intracellular ATP
and ADP concentrations. The Km values for uridine-5'-diphosphoglucose were always 2 to 3 times lower than the intracellular
uridine-5'-diphosphoglucose
concentrations. These results sug
gest that: (a) in these tumor cells, glycogen is essentially synthe
sized by glycogen synthase b via an allosteric activation by
intracellular Glc-6-P; (b) there is no obvious growth-related con
trol of glycogen synthase activity; and (c) the activity of glycogen
phosphorylase seems to be growth dependent with maximal
phosphorylase a activities associated with the period of high
division rate.
INTRODUCTION
Over the last decades, a good deal of attention has been
focused on the modifications in carbohydrate metabolism which
are associated with the neoplastic process. These include: a
higher rate of aerobic glycolysis (14, 30, 31, 48, 50) and the
resurgence of fetal-type isoenzymes (41, 45, 49), as well as an
increase in the rate of glucose transport and its consumption
(14, 15, 51). Neoplasia is also known to be accompanied by
changes in glycogen metabolism when the tumors arise from
tissues which normally store glycogen, e.g., hepatomas (21, 22,
25, 40, 49, 50), choriocarcinomas (12), or the cervix carcinoma
cell line HeLa (1,11).
Much less is known about glycogen metabolism in tumors
originating from epithelial tissues which normally do not store
much glycogen (colon, kidney, lung, etc.). We were able to show
that high amounts of glycogen were stored in colon tumors as
compared to the normal tissue (38). Follow-up studies on more
than 60 cultured human tumor cell lines originating from different
tissues which normally do not store glycogen confirmed that
their glycogen content was always much higher than in the
corresponding normal tissue (35, 39). Moreover, a 3- to 4-fold
increase of the glycogen concentration was observed after mi
tosis during the Gìphase of synchronous cultures (35). In
asynchronous cultures, the glycogen concentration remained at
a constant level during the exponential phase of growth and then
increased 3- to 10-fold when the rate of cell division declined
and during the stationary phase (35, 39, 43). The glycogen
concentrations attained in this way can be considerable. In some
cell lines, they exceed 1 mg per mg of protein (39), a concentra
tion which is even higher than the maximal values observed in
the normal liver. The glycogen level of these cancer cells appears
to be an intrinsic characteristic of each individual cancer: values
during the exponential phase of growth of the cultured cells were
similar to those in the corresponding tumors developed in nude
mice (36).
These data suggest that the neoplastic process leads to major
modifications in the glycogen metabolism of these cells and,
furthermore, that these changes are involved in the process of
their growth. Any further understanding into why glycogen me
tabolism should be involved in both the neoplastic transformation
and the growth of such malignant cells implies a better knowl
edge of how this metabolism is regulated in these cells. There
fore, the purpose of the present work was to investigate the
activity of the key enzymes implicated in this regulation, namely,
glycogen synthase (EC 2.4.1.11) and glycogen phosphorylase
(EC 2.4.1.1), in order to answer 2 questions: (a) how are the
activities of these enzymes related to the variations of glycogen
concentration observed during the growth of these cells? and
(b) how can the activity of these enzymes account for the unusual
glycogen concentrations found in these cells? Since the glycogen
content varies from one cell line to another (39), 3 different cell
lines of different glycogen levels were selected for this study.
MATERIALS AND METHODS
Materials
1Supported by INSERM CRL 79-5-486-7, CRL 79-1-476-7,
106.
2 To whom requests for reprints should be addressed.
Received December 27, 1982; accepted October 4,1983.
154
and ATP 74-79-
The following compounds were obtained from the indicated sources:
UDP-[14C]glucose (uniformly labeled in the glucose moiety) and [14C]
glucose 1-phosphate, from New England Nuclear (Boston, Mass.); GlcCANCER
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VOL. 44
Glycogen Metabolism in Tumor Cells
6-P3 dehydrogenase,
NADP, NAD, UDP-GIc dehydrogenase,
Kit 139.084
for lactic acid determination, phosphoenol pyruvate, pyruvate kinase,
and ATP bioluminescence CLS Kit 567.736, from Boehringer Mannheim
(Mannheim, Germany); and UDP-GIc, Glc-6-P, glucose-1-phosphate, 5'AMP, rabbit liver glycogen, and other chemicals, from Sigma Chemical
Co. (St. Louis, Mo.). Fetal bovine serum and Dulbecco's modified Eagle's
medium were obtained from Grand Island Biological Co. (New York, N.
Y.), and the plastic flasks were from Coming Glass Works (Coming, N.
Y.).
tained in a 200-^1 volume: 40 mw Pipes buffer (1,4-piperazine-diethane
sulfonic acid), pH 6.8; 65 mM NaF; 1% rabbit liver glycogen; and 100 mM
[14C]glucose 1-phosphate (0.02 /iCi/nmol), with or without 1 ITIMcaffeine,
and with or without 1.5 mM AMP, added to 100 n\ of the cell homogenate
supernatants containing 3 mg of protein per ml. The experimental pro
cedure was the same as described for glycogen synthase. Glycogen
synthase and phosphorylase activities are expressed as nmol of glucose
incorporated into glycogen per min per mg of protein. The kinetic
parameters of glycogen synthase were determined at 37°.The ratio of
independence is defined as:
Cell Cultures
Activity without Glc-6-P
The cultured cells used were of established lines derived from human
tumors and were obtained from Dr. JörgenFogh (Sloan Kettering Institute
for Cancer Research, Rye, N. Y.). They included the cell lines: HT-29
(colon carcinoma) (6-8); RT-4 (carcinoma of the urinary bladder) (6, 8,
27, 34); and MeWo (malignant melanoma) (2, 6, 8). The cells were
cultured at 37° in 25-sq cm plastic flasks with Dulbecco's modified
Eagle's medium (25 mM glucose) supplemented with 15% fetal bovine
Activity with 10 mw Glc-6-P
x 100
Extraction of Cell Intermediates: A Comparison of 2 Different Meth
ods
Method 1. The cells were harvested with 0.25% trypsin in 0.6 mM
EDTA. Trypsin was neutralized with fresh serum-supplemented medium
at 4°and the cells were centrifugea as described previously (35, 39).
serum in an atmosphere of 90% air and 10% CO2. All cultures were
mycoplasma free. The glucose consumption was determined at regular
time intervals after medium changes by the glucose oxidase technique
using a BGA2 Beckman glucose analyzer (Beckman Instruments, Gagny,
France). The lactic acid production was determined according to the
enzymatic method of Noll (26).
The cell pellet was rinsed with ice-cold 0.9% NaCI solution and centrifuged. The cell pellet was then stored at -70°. The method was tested
Glycogen and Protein Assays
then immediately frozen by flotation of the flasks on liquid nitrogen and
stored at -70°.
The cells were harvested
centrifugea for glycogen and
39). Glycogen was measured
(46), and the protein content
al. (23).
For the assays, the frozen cell layer (Method 2) was scraped, or the
frozen cell pellet (Method 1) was homogenized in 2 ml of ice-cold 0.5 N
HCICv The mixture was sonicated for 10 sec, kept at 0°for 5 min, and
centrifugea (2°at 4000 x g for 5 min). The supernatant was neutralized
with 5 N KOH and centrifuged (2°at 4000 x g for 5 min). The assays
with 0.25% trypsin in 0.6 mw EDTA, and
protein assays as described previously (35,
with anthrone by the method of Van Handel
was measured by the method of Lowry et
in comparison with the more classical Method 2 because it is more useful
during the early stages of the culture when there is not much material.
Method 2. The culture medium was removed from the flasks, and the
cell layers were rapidly rinsed with ice-cold 0.9% NaCI solution and were
were performed on the neutralized supernatants.
Enzyme Preparation
Freezing and Storage of Organs
The medium was removed from the flasks, and the cell layers were
rapidly rinsed with ice-cold 0.9% NaCI solution and then immediately
frozen by flotation of the flasks on liquid nitrogen; storage was at -70°.
For the assays, the cell layers were thawed in ice-cold homogenization
buffer in a volume adjusted to give approximately 3 mg of cell protein
per ml. The homogenization buffer contained: 62.5 rriM glycylglycine
buffer, pH 7.4; 6.25 rriM EDTA; 125 mw NaF; 0.5 M saccharose; and 5
HIM dithiothreitol. The cells were scraped into the buffer and then
disrupted at 0°,by 20 passages through a 26-gauge needle fitted to a
1-ml polypropylene syringe within a 30-sec time lapse. The homogenates
were centrifugea (15 min at 4°;2500 x g), and the supernatants were
used for the enzyme assays.
Enzyme Assays
Glycogen synthase activity was determined by following the incorpo
ration of glucose from UDP-[14C]glucose into glycogen according to the
method of Thomas ef a/. (44). The standard assay mixture contained in
a 200 n\ volume: 40 mw Tris-HCI buffer, pH 7.8; 4 mw EDTA; 8 rriM NaF;
1% rabbit liver glycogen; and 10 mw UDP-[14C]glucose (0.02 ^Ci/mol),
with or without 10 ITIMGlc-6-P, added to 100 n\ of the cell homogenate
supernatants containing 3 mg of protein per ml. After incubation at 25°,
a 50-¿tlaliquot was spotted onto a filter paper, frozen in a liquid nitrogen
atmosphere, and rinsed in a 66% ethanol bath at 4°to remove free
Four normal mice were killed by cervical dislocation between 10 and
11 a.m. Liver and muscle were excised immediately after death, divided
into small fragments, and frozen immediately in liquid N2 (<1 min after
death). They served as references for Glc-6-P and UDP-GIc concentra
tions. For the assays, homogenization
as described above.
and neutralization were performed
Measurements of Cell Intermediates
ATP and ADP. Aliquots of the neutralized supernatants obtained from
the 2 extraction methods described above were immediately diluted 10fold in a 0.1 M Tris, 2 mM EDTA buffer, pH 7.75. ATP and ADP
measurements were performed according to the method of Spielman ef
a/. (42), using the ATP bioluminescence CLS kit and a LKB 1251
luminometer (LKB Instruments, Orsay, France). For ADP determination,
phosphoenol pyruvate and pyruvate kinase were added as described by
Spielman ef al. (42).
Glc-6-P Concentration. Glc-6-P concentration was measured with an
adaptation of the method of Lang and Michal (19). Fifty n\ of the
neutralized supernatants of cancer cell extracts or normal liver and
muscle were added to 600 ¿dof incubation mixture containing 0.2 M
triethanolamine, pH 7.6; 10 mw MgCfe; 0.8 mM NADP; and Glc-6-P
labeled UDP-GIc molecules. The ethanol bath was renewed 3 times and,
dehydrogenase (commercial solution), 4.5 units/ml. The reaction pro
ceeded over a 15-min period at 25°,after which the reduced NADP was
after 6 to 10 hr, the filter papers were dried and counted. Glycogen
phosphorylase activity was determined according to the method of Wang
and Esmann (47) by following the incorporation of glucose from [14C]
measured in a fluorometer with a 360-nm filter for excitation and a
Vitatron U-11 secondary filter. Taking into account that the cancer cells
studied here possess a very active endogenous Glc-6-P dehydrogenase,4
glucose 1-phosphate
each cell extraction was paralleled with another one in which 10 mM Glc6-P were added during the homogenization in HCIO4. Recovery of all of
the added Glc-6-P ensured that the Glc-6-P dehydrogenase was de-
3 The abbreviations
5'-diphosphoglucose.
JANUARY
into glycogen. The standard assay mixture con-
used are: Glc-6-P, glucose 6-phosphate;
UDP-GIc, undine
* M. Rousset, unpublisheddata.
1984
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155
M. Rousset et al.
stroyed during the extraction and that the observed values for Gte-6-P
were accurate.
UDP-Gic Concentration.
UDP-Gic concentration
was measured with
an adaptation of the method of Keppler and Decker (16) in the same
neutralized supernatants used for Glc-6-P determinations. One hundred
u\ of supernatant were added to 600 pi of incubation medium containing
0.4 M glycine buffer, pH 8.7; 3 mM NAD; and UDP-Gic dehydrogenase
(commercial solution), 30 munits/ml. After a 20-min incubation at 25°,
the reduced NAD was measured in the same way as already described
for Gte-6-P.
The intracellular concentrations of the metabolites (Gte-6-P and UDPGte) took into account the intracellular water content which was appre
ciated by the technique of Reitzer et al. (33). The values (/il/mg of cell
protein) found were: 4.8 ±0.3 (S.D.) for HT-29; 6.9 ±0.8 for RT-4; and
6.8 ±0.8 for MeWo. They were constant during the entire culture period
for all the cell lines.
phase varies from one cell line to another (2-fold for HT-29, 9fold for MeWo, and 3-fold for RT-4).
The glycogen concentrations found in the exponential and
stationary phase do not depend on the glucose consumption
and lactic acid production (Table 1). For instance, HT-29 and
MeWo cell lines, which have similar high glucose consumption
rates and lactic acid production, exhibit different glycogen levels
in the exponential phase and even more pronounced differences
in the stationary phase. A small decrease is observed in the
glucose consumption rates in the transition from the exponential
to the stationary phase in all 3 cell lines. The ratio between lactic
acid production and glucose consumption remains constant,
however, during the entire culture period. The reason why the
lactic acid production in HT-29, MeWo, and RT-4 cell lines should
be slightly higher than the values calculated from their glucose
consumption may be due to the fact that not all of their lactic
RESULTS
Cell Growth and Glycogen
Levels.
The growth-related
changes in the glycogen concentration of the 3 cancer cell lines
are reported in Chart 1a. The glycogen concentration is always
lower and stable during the first, as opposed to the last, days in
culture. This concentration, which is specific for each cell line
(39), begins to increase as soon as confluence is reached (on
Day 5 for MeWo, and on Day 8 for HT-29 and RT-4 cell lines)
and then regularly increases as the rate of cell division decreases
as well as during the stationary phase. It must be noted that: (a)
even in HT-29, which is the cell line in the present series with
the lowest glycogen concentration, this concentration is at least
10 times higher than in the normal tissue (38); (b) the increase in
the glycogen concentration from the exponential to the stationary
HT-29
Tabte!
Glucose consumption and lactic acid production during the exponentialand
stationary phases of growth in culture of the cell lines HT-29, MeWo,and RT-4
protein)Ce«
lineHT-20
Glucose consumption
Gimol/hr/mg
phase*0.60
acid production
protein)Exponential
(/imol/hr/mg
phase"0.46
phase1.20
phase1.05
±0.10°
±0.08
±0.20
±0.10
MeWo
0.73 ±0.15
0.60 ±0.10
1.50 ±0.20
1.30 ±0.15
RT-4Exponential
0.40 ±0.05Stationary
0.30 ±0.05Lactic 0.90 ±0.10Stationary
0.70 ±0.10
" Results are from the fifth day of growth,
Three flasks were used for each
determination.
" Results are from the 18th day of growth,
Three flasks were used for each
determination.
c Mean ±S.D.
RT-4
MeWo
.SO 4.
.500
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_
x
O 200.
100.
.X
LSO
25.
a.
•
o
Z
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£
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O
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s-
-I
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-50 10-
O
«Vj.
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rt_
io
IB
DAYS
Chart 1. Glycogen content and activities of glycogen phosphoryiase and synthase during the growth in culture of the tumor cell lines HT-29, MeWo, and RT-4.
Measurements were performed on Days 1, 2, 3, 5, 7, 9, 12, 15, and 18, at 18 hr after the medium changes, a, protein content (mg) per flask (A) and glycogen
concentrations (•)
(»jg/mg
protein), b, glycogen phosphoryiase activities are reported without (D) or with (•)
1 mM AMP. The activity is expressed as nmol of product
formed per min per mg of protein after 10 min of incubation at 25°(left). The activity ratio is defined as the activity without AMP/activity with 10 mw AMP, the results
being multiplied by 100 (O, right), c, glycogen synthase activities are reported without (V) or with (T) 6 mmGlc-6-P.The activity is expressed as nmol of product formed
per min per mg of protein after 30 min of incubation at 25°(left). The activity ratio is defined as the activity without Glc-6-P/activity with 10 mMGlc-6-P, the result being
multiplied by 100 (O. right). Values,average of 3 different cultures.
156
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VOL. 44
Glycogen Metabolism in Tumor Cells
Tables
Intracellular ATP and ADP concentrations and ADP/ATP ratios in each cell line using 2 different extraction
methods
proteinATPHT-29
nmol/mg of
(%)Method
1a24.0±1.4Õ>'C
15.0
25.3
121
±0.8C
±0.3
±0.3
MeWoRT-4Method
17.0 ±0.9
31.0 ±3.1
3.1 ±0.1
4.4 ±0.1
1616TP
30.5 ±0.2Methods829.7
43.0 ±0.5ADPMethod
5.0 ±0.1Method 7.5 ±0.2ADP/AMethod
218
15
17
See "Materials and Methods.'
6 Mean ±S.D.
c Each value represents the average of 2 flasks. Assays were conducted in duplicate. Cells were harvested
on the fifth day of growth.
acid is derived from glucose. A similar observation has been
described in other cell lines (33).
Glycogen Phosphorylase Activities. The results of glycogen
phosphorylase activities are presented in Chart 10. The total
phosphorylase activity (a + b) was assayed in the presence of 1
HIM AMP, which is a concentration ensuring maximal activation.
Indeed, throughout the culture period, the respective Ao.5AMP
values are 0.06 M for MeWo, 0.125 HIM for HT-29, and 0.2 ITIM
for RT-4 cell lines. A similar pattern of the total enzyme activity
(a + b) is observed in the 3 cell lines, namely, a regular increase
of the a + b activity during the time course of culture. The
observed total activities are different from one cell line to another
and are not directly correlated to the specific glycogen level of
each cell line. In the 3 cell lines, the phosphorylase a activity
exhibits a transitory increase during the first days of culture,
when the cells are highly dividing and the glycogen levels are at
their minimum. Analysis of the activity ratio, which reflects the
phosphorylation state of the enzyme, shows that maximal phosphorylation always occurs during the period of high division rate
in all the cell lines. A progressive decrease in the phosphorylation
state is observed when the rate of cell division declines at
confluency and during the stationary phase (Chart 1b). The
assays were all performed without or with 1 HIM caffeine in the
assay system; the results obtained were the same.
Glycogen Synthese Activities during Cell Growth. As shown
in Chart 1c, the glycogen synthase activity assayed in the
presence of 6 mM Glc-6-P, i.e., the a + b forms of the enzyme,
regularly increases during the culture period, reaching a plateau
between Days 12 and 18. The specific activity of glycogen
synthase a + o varies from one cell line to another and, as in the
case of the glycogen phosphorylase, levels were not related to
glycogen contents. None of the 3 cell lines has more than trace
amounts of the glycogen synthase a form at any stage of the
culture period, not even when glycogen accumulates.
Cellular ATP and ADP Levels. The cellular ATP and ADP
levels were compared for each cell line using 2 different extrac
tion methods. The results are reported in Table 2. Although the
recovery was sometimes higher using Method 2, the ADP and
ATP levels and, more particulariy, the ADP/ATP ratios obtained
by both methods were very similar. The values reported here are
similar to those already reported by other authors (32, 33).
Kinetic Constants for Glc-6-P of Glycogen Synthase b.
Glycogen synthesis can be due to the allosteric activation of
glycogen synthase b by Glc-6-P (20). For this reason, the intracellular Glc-6-P concentration as well as the activation constant
of the enzyme were determined at different stages of culture in
the 3 cell lines. The Ao.5Glc-6-P values on Days 5,9,12,15,
and
JANUARY
Table 3
AO5 Glc-6-P values and corresponding intracellular Glc-6-P concentrations during
the growth in culture of the cell lines HT-29, MeWo, and RT-4
Glc-6-PCell
1*1.812.5
(HIM)
Method
lineHT-29MeWoRT-4Day591215185912151859121518Ao5a
2o5.0
1.2e3.34.51.60.81.2
5.4 ±
0.8e2.2
±
±0.51.22.511.31.5
2.8
0.72.3
±
±0.31.52.5Method
2.7
±0.6
AOSGlc-6-P values were obtained from the corresponding Lineweaver-Burk
plots.
See 'Materials and Methods."
0 Mean ±S.D. of the Glc-6-P concentrations obtained on Days 5,9,12,15,
and
18. For each day, 2 different flasks were used, and the assays were conducted in
triplicate. These Glc-6-P concentrations were determined on the same cultures
which were used for the determination of the A« Glc-6-P values.
18 of each cell line were calculated from the corresponding
Lineweaver-Burk plots. An increase of the Ao5Glc-6-P values is
observed during the culture period: a similar pattern is found in
all 3 cell lines (Table 3). Table 3 also records the corresponding
intracellular Glc-6-P concentrations obtained by both extraction
methods. No clear-cut change in Glc-6-P levels was found during
the culture period. The Glc-6-P concentrations range from 2.2 to
5.4 HIM and were similar for each cell line in the 2 types of
extracts. They are 6- to 10-fold higher than those obtained in
normal liver and muscle assayed under the same conditions.
These values are, respectively, 0.34 ±0.05 mM for the liver and
0.66 ±0.07 ITIMfor the muscle, and are very similar to those
already reported in normal liver and muscle by other authors
(52). The Glc-6-P concentrations in the cancer cell lines are of
the same order of magnitude as the calculated A0;,Glc-6-P values
for each cell line.
Kinetic Constants for UDP-GIc of Glycogen Synthase b. The
Km values were measured in the presence of 6 PIM Glc-6-P as
permanent allosteric activator. They were calculated on Days 5,
9, 12, 15, and 18 of the cell cultures from the corresponding
Lineweaver-Burk plots. As summarized in Table 4, the Kmvalues
always increase gradually over the time course of the culture.
Table 4 gives the corresponding intracellular concentrations of
1984
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157
M. Roussel et al.
UDP-GIc. Similar values were obtained for each Å“il line in the 2
types of extracts. These values are fairly constant during the
culture and are found to be at least twice as high as the
corresponding Km values. In the case of the HT-29 and MeWo
cell lines, a curvilinearity of the Lineweaver-Burk plots was
observed on Days 15 and 18 (Chart 2). The UDP-GIc concentra
tions in these cancer cell lines are at least 10-fold higher than
those in normal liver or muscle assayed in the present report.
The values obtained are 0.32 ±0.03 ÕTIM
for the liver and 0.06
±0.02 rnw for the muscle, these levels being very similar to
those already reported by other authors (52).
Test of Conversion of Glycogen Synthase b to a Form.
Glycogen synthase in crude extracts of the 3 cultured cell lines
is predominantly in the Glc-6-P-dependent form (b form) through
out the culture period. In order to check whether such a situation
was due to an impairment of the dephosphorylating mechanism
of the enzyme in the same way as has been reported for other
models (13), the crude extracts were incubated without NaF.
The results reported in Chart 3 show that this incubation resulted
in a rapid partial conversion of synthase b to synthase a (within
15 min), with the total activity of a + b forms remaining constant.
The ratio of independence was increased in the 3 cell lines, but
the values reached in the cell extracts from the last stages of
Tabte4
K„
valuesfor UPD-GIcand corresponding intracellular UDP-GIcconcentrations
during the growth in culture of the cell lines HT-29, MeWo, and RT-4
HT-29
UDP-GIc
MeWo
Method 2*
Method!
HT-29MeWoRT-45912151859121518591215180.460.550.55
Cell line
Day
K,,,'1(mu)
0.8C0.550.590.30.350.42
4.3 ±
0.72.5
±
75 min
RT-4
0.50.50.50.50.770.77
2.3 ±
0.83.3
±
0.31.051.354.0 2.9 ±
±0.6
" Kmvalues were obtained from the corresponding Lineweaver-Burkplots.
See "Materials and Methods."
0 Mean ±S.D. of the UDP-GIcconcentrations obtained on Days 5, 9, 12, 15,
and 18. For each day, 2 different flasks were used, and the assays were conducted
in triplicate. These UDP-GIcconcentrations were determined on the same samples
used for the determinations of the Gfc-6-Pconcentrations.
Chart 3. Conversion of glycogen synthase b to synthase a. Extracts of cells
from Days 5 (•)
and 18 (•)
were incubated at 37°without NaF to see if a
dephosphorylationof glycogen synthase could occur. At the indicated times (0,15,
45, and 75 min), 8 mm NaF was added, and glycogen synthase activity was
assayed. This activity was determined with or without 10 mMGfc-6-Pas described
before. The ratio of independence(see "Materials and Methods") is reported each
time. Cu/ves, from one experiment.
>V-\fe
Vv-Vo
HT-29
0.2.
Chart 2. Effect of UDP-GIcon the activity of
glycogen synthase b. Enzyme was assayed
with 6 HIMGte-6-P and various UDP-GIccon
centrations on Days 5 (•),
9 (A), 12 (O), 15 (D),
and 18 (•)
at 37°.Curves, from 2 separate
cultures.
0.1.
V [UDP-GIc]
158
(mM-1)
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VOL. 44
Glycogen Metabolism in Tumor Cells
the cultures were always lower than those in the Å“il extracts
from earlier growth phases.
reflect a heterogeneity of the enzyme extracts at these stages
of the culture. However, both the values of Ao 5Glc-6-P and those
of Kmfor UDP-Glc are compatible with the synthesis of glycogen
DISCUSSION
via the phosphorylated forms of the enzyme during the whole
culture period in the 3 cancer cell lines studied.
As the ratio between glucose consumption and lactic acid
production is constant in each cell line during the entire culture
period and, furthermore, the Glc-6-P and UDP-Glc concentrations
The unusual accumulation of glycogen in human cultured
cancer cells and its growth-related variations prompted this
investigation on the way glycogen metabolism is regulated in
these cells. In mammalian tissues, this is known to be essentially
mediated through the covalent modifications of glycogen synthase and phosphorylase by phosphorylation and dephosphorylation via an integrated control (3, 5,10,17, 20).
The results presented here show that both of the enzymes,
glycogen synthase and phosphorylase, are present in the 3
cancer cells studied. The pattern of these enzyme activities as a
function of the pattern of glycogen accumulation during cell
growth suggests, however, that the mechanism(s) of control of
glycogen synthesis in these cancer cells is different from the
one(s) attributed to normal cells. One divergence stems from the
fact that the glycogen synthase is almost completely in a phosphorylated, Glc-6-P-dependent form throughout the entire cul
ture period. Even the considerable increase of glycogen accu
mulation in the last stages of the culture of the MeWo cell line is
not correlated with any conversion of glycogen synthase to the
active form. Such a situation is not due to an impairment of the
dephosphorylating mechanism of the enzyme, since it is possible
to obtain a conversion to the dephosphorylated form in cellular
extracts incubated without NaF. Since the structure of glycogen
is normal in these cancer cell lines,4 it can be assumed that
glycogen synthesis proceeds normally via the branching enzyme
and glycogen synthase. Consequently, glycogen synthesis in
these cells may only be achieved by a phosphorylated, Glc-6-Pdependent form of glycogen synthase. It is generally agreed that
this form of the enzyme is inactive under normal physiological
conditions (29). However, the 3 tumor cell lines studied all
contained Glc-6-P concentrations high enough to result in an
allosteric activation of the phosphorylated forms of the glycogen
synthase which is present throughout the culture period. Such
high Glc-6-P concentrations were obtained by 2 different extrac
tion methods and cannot be due to an artifact of the preparations
as controlled by ATP and ADP concentrations in the same
extracts. The 3 tumor cell lines studied contained much higher
concentrations of intracellular Glc-6-P at all stages of the culture
than those usually reported in normal cells (9,12) or those found
in normal liver and muscle either in the present study or by other
authors (52). The reason why such high Glc-6-P concentrations
should be present in these human cancer cells still needs to be
investigated. Such increase in Glc-6-P concentrations has al
ready been reported in rat tumor cells or transformed cells as
compared to normal cells (4). One possible explanation is that
cancer cells contain a hexokinase which is not appreciably
inhibited by Glc-6-P (28). The total glycogen synthase activity
increased as the time in culture was extended. This enhancement
was found in all 3 cell lines and was always accompanied by an
increase in the Ao 5Glc-6-P values and a decrease in affinity for
UDP-Glc. Further studies will be needed to investigate whether
new forms of phosphorylated glycogen synthase appear at the
transition from the exponential to the stationary phase of growth.
There is some evidence to suggest that this may be the case: a
curvilinearity of the Lineweaver-Burk plots is observed on Days
15 and 18 in the HT-29 and MeWo cell lines, which may well
JANUARY
1984
are likewise constant, it is most unlikely that the variations of
glycogen accumulation, which are related to the different growth
phases, can be explained by clear-cut modifications in glycogen
synthesis. These changes can be more satisfactorily attributed
to the differences in the phosphorylation state of glycogen phos
phorylase. Indeed, the phosphorylation state of this enzyme was
found to vary significantly with the growth-related pattern of
glycogen accumulation. Thus, maximal activations of glycogen
phosphorylase were always associated with the lowest glycogen
levels during the periods of cell division, whereas a progressive
inactivation of this enzyme regularly occurred when the rate of
cell division declined and glycogen levels increased. Further
studies on glycogen turnover should establish whether glycogen
synthesis remains constant during the whole culture period. If
so, one could envisage that the lower values found during the
period of high division rate could result from the activation of
glycogen phosphorylase, while the progressive accumulation of
glycogen could result from a progressive inactivation of this
enzyme when the cells stop dividing. This pattern of variations
in the phosphorylation state of glycogen phosphorylase during
cell growth, and especially the association of an activation of the
enzyme with the period of cell division, would suggest that the
phosphorylation of glycogen phosphorylase is related to the
process of cell division. Further studies would need to investigate
whether some effectors could be involved in the dual control of
cell division as well as of the known enzymatic cascade leading
to the activation of glycogen phosphorylase. It is already known
that HT-29 cells have an adenylate cyclase-mediated control
system of glycogenolysis (18, 24, 37).
Taken together with previous results on glycogen accumula
tion in human malignant epithelial cells (35, 38, 39), the present
data strongly suggest that glycogen metabolism may play an
important role in the physiology of cancer cell division. With
regard to the results already reported of the occurrence of a
peak of glycogen accumulation in the middle of the G, phase of
the cell cycle (35), one of the questions which remains to be
resolved is whether, in such cancer cells, glycogen use, resulting
from the activation of glycogen phosphorylase, is a prerequisite
or a consequence of DMA synthesis.
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RESEARCH
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VOL.
44
Growth-related Enzymatic Control of Glycogen Metabolism in
Cultured Human Tumor Cells
Monique Rousset, Hervé Paris, Guillemette Chevalier, et al.
Cancer Res 1984;44:154-160.
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