[CANCER RESEARCH 33, 2402 2407, October 1973]
Incorporation of 2-Deoxy-D-glucose into Glycoproteins of Normal
and Simian Virus 40-transformed Hamster Cells1
Sheldon Steiner,2 Richard J. Courtney, and Joseph L. Melnick
Department of Virology and Epidemiology. Baylor College of Medicine, Houston, Texas 77025
SUMMARY
2-Deoxy-n-glucose, which was regarded as being poorly
metabolized by animal cells and therefore useful for trans
port studies, is incorporated rapidly and intact into glycoprotein. The polyacrylamide gel profile of the incorporation
of 2-deoxy-D-glucose into glycoprotein parallels that of
glucosamine and fucose. The incorporation of 2-deoxy-Dglucose into glycoprotein and glycolipid may help to explain
several of the inhibitory features of 2-deoxy-D-glucose on
virus-mediated events. Profiles of the glycoproteins ob
tained by polyacrylamide gel electrophoresis reveal that the
simian virus 40-transformed cells have a reduced amount of
a high-molecular-weight glycoprotein as compared to nor
mal cells.
INTRODUCTION
The glucose analog DGLC3 is being used extensively in
studies of sugar transport in normal and virus-transformed
cells (15, 17, 21, 25, 30), as a metabolic inhibitor of cultured
cells (3, 11) and of cells transformed by virus ( 14) as well as
an inhibitor of envelope biosynthesis of a number of RNA
viruses (18). In animal cells, it has been assumed that
DGLC is not metabolized beyond deoxyglucose 6-phosphate (15, 19, 25, 29) or phosphodeoxygluconic acid (12).
However, earlier studies with yeast had shown that, in
addition to being phosphorylated, DGLC is converted to
uridine diphosphodeoxyglucose (5, 16), guanosine diphosphodeoxyglucose (6), and deoxygluconic acid (4). In more
recent studies with yeast, Steiner and Lester (31) showed
that DGLC is further incorporated into glycolipid, and
Biely et al. (7) demonstrated its incorporation into yeast cell
wall mannan. These observations led us to investigate the
metabolic fate of DGLC in mammalian cells; recently, we
reported on its incorporation into the glycolipids of normal
and SV40-transformed hamster cells (32).
In the present paper we report on the incorporation of
DGLC into the glycoproteins of normal and SV40-trans'This investigation was supported in part by Research Contract NO 1
CP 33257 within the Virus Cancer Program of the National Cancer Insti
tute, NIH, Bethesda, Md., and by Research Grant ACS-IN-27M from
the American Cancer Society.
"Recipient of Special Fellowship Award I-F3-CA54.999 from the
National Cancer Institute, NIH.
3The abbreviations used are: DGLC, 2-deoxy-D-glucose; SV40, simian
virus 40.
Received October 9, 1972: accepted June 18, 1973.
2402
formed hamster cells. In our previous work with yeast (31)
we showed that DGLC is not degraded during its metabo
lism. The same applies in mammalian cells, and therefore
the usefulness of DGLC as a nonrandomized precursor of
the glycoproteins of virus-transformed cells was also ex
plored.
MATERIALS
AND METHODS
Materials. Radiochemicals were obtained from the fol
lowing sources: DGLC-1-14C (53.5 mCi/mmole) and gen
erally labeled DGLC-3H (7.9 Ci/mmole) from New Eng
land Nuclear, Boston, Mass.; glucosamine-1-MC (55 mCi/
mmole), glucosamine-6-3H (14.45 Ci/mmole), and fucose1-3H (1.8 Ci/mmole) from Amersham Searle, Arlington
Heights, 111., uniformly labeled leucine-14C (310 mCi/
mmole) and leucine-4,5-3H (15 Ci/mmole) from Schwarz/
Mann, Orangeburg, N. Y. The purity of the DGLC-3H and
DGLC-MC was examined by cochromatography with au
thentic DGLC-12C in each of these 3 solvent systems: 1-butanol:pyridine: H2O (6:4:3, v/v), l-butanol:ethanol: H2O
(50:32:18, v/v), and 1-butanol:acetic acid:H2O (4:1:5,
v/v) followed by the use of autoradiography and a non
specific sugar spray (27) (for the carrier DGLC-12C). The
results indicated that both the DGLC-14C and DGLC-3H
were pure.
Normal hamster embryo fibroblast cells and SV40-transformed cells (H50 line) (I) were grown in Eagle's medium
supplemented with 10% fetal calf serum and antibiotics. The
possible presence of pleuropneumonia-like organisms was
excluded by the use of immutiofluorescence, electron mi
croscopy, and subculturing-on pleuropneumonia-like orga
nism liquid and solid .medium (Grand Island Biological
Corp., Grand Island, N. Y.). Cells were routinely seeded at
7 x IO5cells/16-oz bottle.
Fractionation of Cells. Cell monolayers were dislodged
from the glass surface by gentle scraping into Tris-buffered
NaCl solution. The resulting cell suspension was subjected
to one of the following treatments: (a) repeated extraction of
the particulate material with 0.5 \ perchloric acid or 0.5 N
trichloroacetic acid as described by Ward and Plagemann
(33); or (b) disruption in a Dounce homogenizer and
centrifugation at 135,000 x g for 30 min to sediment the
particulate material which was then washed with water.
Delipidation of samples was carried out by extracting twice
with CHC13:CH3OH
(2:1, v/v) and twice with
CHC13:CH3OH (1:2, v/v), based on the method of Folch
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VOL. 33
Deoxyglucose
Incorporation
into Glycoproteins
determined as seen in Table 2. The radioactivity found in
the delipidated, acid-insoluble residue of normal and trans
formed cells represented 3.3 and 2.4%, respectively, of the
total cellular radioactivity, while the radioactivity in the
lipid fraction accounted for 13.4 and 15.8%, respectively.
Since the usefulness of DGLC as a measure of glycoprotein
metabolism in mammalian cells depends on whether it
remains intact, the fate of the incorporated radioactivity
was examined. Delipidated paniculate material from nor
mal and SV40-transformed cells incubated for 20 min or for
48 hr with DGLC-14C or DGLC-3H was subjected to mild
acid hydrolysis and chromatographed in System 1. The
results seen in Tables 3 and 4 show that approximately 90%
of the protein-associated radioactivity was DGLC. No trace
of radioactivity was found in other natural sugars. The same
results were obtained from hydrolysate chromatographed in
Solvent System 2 and from the hydrolysate of 14C-labeled
material chromatographed in 2 dimensions.
To determine whether the DGLC radioactivity that was
observed in the acid-insoluble fraction and therefore be
lieved to be glycoprotein was in fact such, whole paniculate
cells or a delipidated, acid-insoluble fraction from cells
labeled with DGLC-3H and leucine-14C were subjected to
polyacrylamide gel electrophoresis. The profile of wholecell paniculate material from normal and transformed cells
(Chart 1) reveals that the peaks of DGLC-3H coincide with
peaks of leucine-l4C-containing material. In addition, the
acid-insoluble, delipidated fraction from cells labeled with
DGLC-3H for 20 min (Chart 2) exhibited similar peaks of
radioactivity typical of glycoproteins.
A comparison of the polyacrylamide gel electrophoresis
profiles of whole-cell paniculate material from normal and
transformed cells labeled both with DGLC-3H and glucosamine-14C was also made. The polyacrylamide gel electro
phoresis profiles of normal and transformed cells (Chart 3)
revealed that the glucosamine and DGLC radioactivity had
virtually the same pattern, further substantiating our evi
RESULTS
dence that DGLC is incorporated into cellular glycoprotein.
A comparison of the normal cell glycoprotein profile with
It is known from the work of others (15, 17, 24) that that from transformed cells revealed reduction in the
DGLC is readily taken up by normal, SV40-transformed
relative amount of radioactivity in the Peak 1 area of the
hamster cells and Novikoff hepatoma cells in short-term
transformed cell (Charts 1 and 3). For further study of this
labeling experiments. Work from this laboratory (32) difference in labeling, normal cells were grown in medium
showed that hamster cells exposed to DGLC-MC for 48 hr supplemented with glucosamine-3H and transformed cells
incorporated the intact sugar into glycosphingolipids of
hamster cells. Hence, we sought to examine other aspects of
Table 1
the metabolism of DGLC, such as its incorporation into
Distribution a/ radioactivity in normal and SV40-lransformed
cells
glycoprotein after short- and long-term labeling periods.
pulsed for 20 min with DGLC-3H
Normal and SV40-transformed cells were incubated in
phosphate-buffered saline supplemented with DGLC-3H for
Cellular fraction
20 min, and the distribution of radioactivity in the acid-solu
Acid-insoluble Acid-soluble
% acidble and acid-insoluble fractions was measured (Table 1). Of
cpm x 10~3 cpm x 10~3 insoluble cpm"
Cell type
the total DGLC-3H incorporated, approximately equal
amounts (3.2 to 3.6%) were found in the acid-insoluble
3.64
3.069
116
Normal
material from normal and SV40-transformed cells. Previ
SV40-transformed
3.24
5.343
179
ously, we had found that approximately 15% of the
" Normal and SV40-transformed cells were incubated in phosphateradioactivity was found in lipid after a 30-min labeling buffered saline supplemented with DGLC-3H (1.27 x 10'3 mM) for 20
period (32). The distribution of radioactivity in acid-soluble, min. The average of 2 experiments is presented. No more than 10 to 15%
acid-insoluble, and lipid-soluble fractions from cells grown of the total acid-insoluble radioactivity above was extractable with organic
for 48 hr in medium supplemented with DGLC-3H was also solvents (i.e.. Folch extraction).
(13). Solubilization of samples for polyacrylamide gel
electrophoresis was accomplished by heating an aliquot for
2 min at 100°in a mixture of sodium dodecyl sulfate, 1.0%;
urea (5.0 vi), 1%; and 2-mercaptoethanol, 1%.
Polyacrylamide Gel Electrophoresis. The method used for
preparation and slicing of 6.6% polyacrylamide gels has
been previously described (9). A 10% solution of BioSolv
(Beckman Instruments, Palo Alto, Calif.) was used for
elution of the radioactive material from 2-mm-thick minced
gel fractions. After an overnight incubation, 7.0 ml of
scintillation fluid [PPO, 4 g; dimethyl-POPOP, 0.4 g; 100
ml Beckman Bio-Solv (BBS-3) in 900 ml toluene] were
added, and the radioactivity was measured in a Beckman
LS-250 scintillation spectrometer.
Acid Hydrolysis and Identification of Products. Delipidated DGLC-14C or DGLC-3H-labeled paniculate mate
rial, to which were added 4 ¿¿moles
of carrier DGLC, was
hydrolyzed at 100°in 0.05 N H2SO4 for 20 min. The mixture
was neutralized with 0.4 N Ba(OH)2 or with a saturated
solution of BaCO3, and the barium sulfate was removed by
centrifugation. The 3H-labeled hydrolysate was chromatographed as a band on microcrystalline cellulose thin-layer
plates (Quantum Industries, Fairfield, N. J.) in either
l-butanol:pyridine: H2O (6:4:3, v/v), or l-butanol:propionic acid:H2O (6:3:4, v/v). Following chromatography
approximately one-third of the lane was sprayed with either
a nonspecific sugar reagent, p-anisidine phthalate (27), ora
spray more specific for deoxy sugars, HC1O4: acetone (36).
The remainder of the lane was subdivided into small bands
parallel to the origin, scraped into scintillation vials, and
counted. The 14C-labeled hydrolysate was chromatographed
in 2 dimensions using the above solvent systems. The
radioactive material was visualized by autoradiography and
the carrier DGLC was located with either of the sugar
sprays indicated above.
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2403
S. Steiner, R. J. Courtney, and J. L. Melnick
Table 2
Distribution of radioactivity in lipid-soluhle. acid-soluhle. and acid-insoluhle fractions of normal and
SV40-lransformed cells labeled with DGLC-'H for 48 hr
Cells were grown for 48 hr in medium supplemented with DGLC-3H (10 jtCi/ml of medium). Cell
monolayers were washed twice with cold phosphate-buffered saline and scraped into phosphate-buffered
saline. See text lor method of preparation and delipidation of acid-insoluble material.
SV40-transformed cells
Normal cells
total3H
of
total3H
of
radio
radio
310.1139.8262871,902%
3H x 10
35,5335,326207858%
3H x IO
activity86.683.33.213.4cpm
activity84.281.82.415.8
Total
fractiona.
radioactivity in nonlipid
(radioactivity)b.
Acid-soluble
(radioactivity)Total
Acid-insoluble
radioactivity in lipid fractioncpm
Table 3
Mild acid hydrolysis of short-term DGLC-'H labeled material
Preparation of cells, hydrolysis conditions, chromatography (System 1), and measurement of
the 3H radioactivity are described in "Materials and Methods." Standards chromatographed in
parallel lanes in the above system include: DGLC, Rh 0.56: glucose. Rh 0.38; galactose. Rt 0.33;
mannose, Rt 0.42: and fucose, RK0.47.
SV40-transformcd cells
Normal cells
3HRadioactivity
of starting acidof starting acidinsoluble material
insoluble material
recovered as DGLCcpm 3H17,150%recovered as DGLC
cpm
in acid-4,158%
insoluble material
before hydrolysis
Radioactivity following
3,472
hydrolysis, which cochromatographed with
authentic DGLC
90
15,607
91
Table 4
Mild acid hydrolysis of 48-hr deoxyglucose-labeled delipidated paniculate material
Preparation of cells, hydrolysis conditions, chromatography (System I), and measurement of
the 3H radioactivity are described in "Materials and Methods." Standards chromatographed in
parallel lanes in the above system include: DGLC. RK0.56; glucose, RK0.38: galactose, RK0.33;
mannose, Rt 0.42; and fucose, Rf 0.47.
Radioactivity in acid-insoluble
material before hydrolysis
Radioactivity following hydroly
sis which cochromatographed
with authentic DGLC1743
Normal cells
SV40-transformed cells
% of initial counts
cpm 3H recovered as DGLC
% of initial counts
cpm 5H recovered as DGLC
15922184
were grown in medium containing glucosamine-14C. The
cells were mixed prior to solubilization and subjected to
polyacrylamide gel electrophoresis (Chart 4). The profile
reveals that the relative amount of label incorporated into
glycoproteins in the Peak 1 area was reduced in the
transformed cells. Fucose was also used to substantiate this
phenomenon. Unlike glucosamine, fucose is not incorpo
rated into mucopolysaccharide (23), and hence it is a useful
specific marker of glycoprotein metabolism (2). The poly
acrylamide gel electrophoresis profiles of fucose-labeled
2404
91.3
205193.9
paniculate material from normal and transformed cells are
shown in Chart 5. The apparent decrease in the amount of
glycoproteins of transformed cells in the Peak 1 area also
extends to fucose-containing glycoproteins.
DISCUSSION
DGLC, a known inhibitor of transformed cell growth (3,
14), is currently being extensively used in studying several
CANCER RESEARCH
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VOL. 33
Deoxyglucose Incorporation into Glycoproleins
2000
1500
iood
PEAK I
200l3
800
-
50 -
600
400
100
O 200
50 o
o
Ü
0
x
250
O
IO 20 30 40 50
FRACTION NUMBER
O
500
2000
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800
400
600
300
400
200
200
100
io
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ID
O
Ü
O)
o
Ü
O
O
IO 20 30 40 50
FRACTION NUMBER
concentration of the added DGLC. The molecular weight of
nonglycosylated proteins was not affected, leading us to
believe that the effect was on the oligosaccharide portion of
the molecule (10). We reasoned that substitution of DGLC
for the natural sugar results in blockage of chain elongation
of the glycoprotein.
The manifold reports on DGLC transport in normal and
transformed cells were based on the assumption that DGLC
was not metabolized beyond DGLC-6-phosphate or 6-phosphodeoxygluconate (24). The present and a previous report
(32) show that this assumption is not true. After long
periods of labeling, about 16%of the DGLC radioactivity is
associated with protein or lipid. However, the present
observations probably do not negate the bulk of the
implications drawn from the DGLC transport studies. Most
of these have been conducted on cells labeled for 1 hr or less.
We show that after 20 to 30 min most of the DGLC is in
small-molecular-weight material (i.e., acid-soluble radioac
tivity). The major point of the present observations is that
the features of the metabolism of DGLC are similar to those
of other sugars; hence, the use of DGLC in sugar transport
offers few unique advantages.
Another facet of this study was the use of isotopically
labeled DGLC to examine the total cellular glycoproteins of
normal and SV40-transformed hamster cells. In addition,
other well-established sugar precursors of glycoproteins
(i.e., glucosamine and fucose) (8, 22, 24, 28, 34, 35, 37) were
used. The results using all 3 carbohydrates were comparable
in showing that SV40-transformed cells had reduced
amounts of a high-molecular-weight paniculate glycoprotein(s). The results of this study are in agreement with
those of Chiarugo and Urbano (8), who found a reduction in
the amount of a high-molecular-weight glycoprotein on the
surface membrane of polyoma-transformed hamster cells.
These authors suggested that the reduction was due to an
alteration of the overall structure of the membrane that
O
Chart 1. Polyacrylamide gel electrophoresis pattern of whole-cell
paniculate preparation of normal cells (a) or SV40-transformed cells (b)
grown Tor48 hr in medium supplemented with both DGLC-3H (15 juCi/ml
of medium) (O) and leucine-"C (1.5 ^Ci/ml of medium)(•).Details ot the
preparation of the cells lor polyacrylamide gel electrophoresis are de
scribed in the text.
aspects of cellular metabolism and viral replication. Until
recently, it was considered that the effect of DGLC on
cellular growth and metabolism was due to its inhibitory
effect on several enzymes of glucose metabolism (3).
However, the recent work of Hatanaka (14) using low levels
of DGLC corresponding to those used in this study suggest
an alternative explanation. Our work suggests that the
inhibitory effect of DGLC may result from its incorporation
into membrane glycolipid or glycoprotein.
In other experiments (10) in which the effects of DGLC
on herpesvirus infection have been studied, we have shown
that at higher concentrations of DGLC the molecular
weight of the glycoproteins was reduced as a function of the
I
IO
x
600 "i
600
i
tu 400
co
400
200
200
O 2
¡o
—¿
S
co
81
cr
O
IO
20
30
40
FRACTION NUMBER
50
to
Chart 2. Polyacrylamide gel electrophoresis patterns of whole-cell,
acid-insoluble, delipidated material from normal (•)and SV40-transformed (O) hamster cells incubated in DGLC-3H (1.27 x IO 3mvi) for 20
min in phosphate-buffered saline at 37°.The data represent a composite of
2 gels. Details of the preparation of cells for polyacrylamide gel electropho
resis are given in the text.
OCTOBER 1973
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2405
5. Steiner,
R. J. Courtnev,
and J. L. Melnick
6000
6000
3000
2500
5000
1500
Peak 3
ü
z
2000
4000
1500
3000
1500
Peak l
1000
5
1000
Peak 4
(O
z
2000
1000
13
O
0
Z)
O
500
0
1000
0
O
o
500
500
10 20 30 40 50
FRACTION NUMBER
l
10
6000
30
FRACTION
6000
3000
1250
5000
»
1000
4000
î
x
IQ
20
50
40
NUMBER
Chart 4. Polyacrylamide gel electrophoresis profile of glucosaminelaheled whole-cell paniculate preparation of normal cells (O) labeled with
glucosamine-3H ( 10¿iCi/mlof medium) and transformed cells (•)labeled
with glucosamine-"C (I nCi/ml of medium). Cells were mixed prior to
solubili¿ation.Further details of the preparation of the cells for polyacrylamide gel electrophoresis are given in the text.
z
750
30005
-
\
o
0
500
2000
250
looo g
"0
10
_L
20
l
30
J_
40
6000
H
z
50o
oe
o
i/i
FRACTION NUMBER
z
<
Chart 3. Polyacrylamide gel electrophoresis profiles of whole-cell
paniculate preparation of normal cells (a) or SV40-translormed cells (/>)
grown for 48 hr in medium supplemented with both DGLC-'H (15
/iCi/ml of medium) (O) and glucosamine-"C (1.5 /iCi/ml of medium)
(•).Details of the preparation of the cells for polyacrylamide gel electro
phoresis are described in the text.
-
Cm FROM
prevented insertion of the glycoprotein. Others (22, 28) have
shown significant variations in the glycoprotein and protein
patterns of the membranes of SV40-transformed
mouse
cells. Although the basis of the alterations in the glycoproteins of transformed cell membranes is not clear, it is
probable that these alterations will be important in assessing
some of the cardinal properties associated with the trans
formed cells. In this regard, the use of DGLC, with its
unique properties as both inhibitor and sugar substitute,
2406
1000 ^
ORIGIN
Chart 5. Polyacrylamide gel electrophoresis pattern of whole-cell
paniculate preparation of normal (O) and transformed cells (•)grown in
medium supplemented with fucose-'H (10 jiCi/ml of medium). The data
represent a composite of 2 gels. Details of the preparation of cells for
polyacrylamide gel electrophoresis are given in the text.
may aid in understanding differences
transformed cell membranes.
between normal and
CANCER RESEARCH
VOL. 33
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Deoxyglucose Incorporation into Glycoproteins
ACKNOWLEDGMENTS
We express our appreciation to Merrel! Charlton for her skillful
assistance.
20.
21.
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2407
Incorporation of 2-Deoxy-d-glucose into Glycoproteins of
Normal and Simian Virus 40-transformed Hamster Cells
Sheldon Steiner, Richard J. Courtney and Joseph L. Melnick
Cancer Res 1973;33:2402-2407.
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