Download Inhibition of L-Fucose Incorporation into

[CANCER
RESEARCH
37, 4250-4255,
December
1977]
Inhibition of L-Fucose Incorporation into Glycoprotein of
Sarcoma 180 Ascites Cells by 6-Thioguanine1
John Stephen Lazo, Kou M. Hwang,2 and Alan C. Sartorelli
Department
Connecticut
of Pharmacology
06510
and Developmental
Therapeutics
Program,
Comprehensive
SUMMARY
The incorporation of [3H]fucose into glycoproteins of
Sarcoma 180 cells in vitro was decreased within 2 hr of
exposure to 6-thioguanine (6-TG); 50% inhibition was pro
duced by 8 /nM 6-TG. Under identical conditions, no de
crease in [3H]glucosamine incorporation into acid-insoluble
material was detected. Similarly, [3H]fucose incorporation,
but not [14C]glucosamine incorporation into glycoproteins
of Sarcoma 180 ascites cells in vivo, was significantly
reduced 1 to 6 hr after 6-TG treatment (20 mg/kg) of mice
bearing 6-day implants of this neoplasm; the maximum
depression of fucose utilization occurred at 2 hr after drug
treatment. The decrease in [3H]fucose incorporation into
glycoprotein was dose dependent and reached a maximum
reduction of 77% of control incorporation 2 hr after 6-TG (10
mg/kg). The radioactivity from fucose found in acid-soluble
extracts of Sarcoma 180 cells was decreased by 45% after
6-TG (10 mg/kg). In contrast, this concentration of 6-TG did
not decrease the level of radioactivity from [3H]fucose found
in acid-soluble extracts of a subline of Sarcoma 180 resist
ant to 6-TG and produced only a 38% decrease in fucose
incorporation into the acid-insoluble fraction of this neo
plasm. The inhibition of [3H]fucose incorporation into gly
coproteins of Sarcoma 180 produced by 6-TG appeared to
be due to a drug-induced decrease in the formation of
guanosine diphosphate-[3H]fucose and a concomitant de
crease in the intracellular content of guanosine diphosphate-fucose. These data suggest that 6-TG exerts, as
a metabolic lesion, a suppression of guanine nucleotide
sugar biosynthesis. Since fucose is largely a terminal car
bohydrate of glycoproteins and glycolipids of the plasma
membrane, 6-TG may alter membrane composition. This
phenomenon may be associated with the cytotoxicity of 6TG to neoplastic cells.
INTRODUCTION
The antimetabolite 6-TG3 is a clinically useful drug in the
treatment of the acute leukemias, largely in combination
with other drugs (7, 22). The available evidence indicates
1 Supported in part by Grants CA-02817 and CA-16359 from the National
Cancer Institute, USPHS.
2 Present address: Section of Immunology, Department of Developmental
Therapeutics,
M. D. Anderson Hospital, University of Texas System Cancer
Center, Houston, Tex. 77025.
3 The abbreviations
used are: 6-TG, 6-thioguanine;
TCA, trichloroacetic
acid.
Received
4250
February 7, 1977; accepted
August 23, 1977.
Cancer Center,
Yale University
School of Medicine,
New Haven,
that the antineoplastic activity of 6-TG requires its conver
sion to the nucleotide form (4). Nevertheless, the precise
metabolic lesion(s) responsible for its cytotoxicity remains
unclear. Several sites of action have been proposed, includ
ing inhibition of purine nucleotide biosynthesis de novo
(23), inhibition of purine ribonucleotide interconversion
(19), inhibition of protein synthesis (9), and incorporation
into RNA and DNA following conversion to the nucleotide
form (17, 18). Although the available evidence favors the
incorporation of 6-TG into DNA as being critical to the
cytotoxic action of this drug (26), the mechanism by which
the incorporated analog exerts its cytotoxic effects has not
been delineated (22), and an exception to the importance
of this lesion to antineoplastic activity exists (24). Conse
quently, it is possible that other biochemical alterations
caused by 6-TG might be involved in its cytotoxic mode of
action.
Previous studies from our laboratory have shown that
the exposure of Sarcoma 180 to 6-TG in vitro results in cell
surface alterations detected by concanavalin A agglutina
tion (11, 12). To gain evidence for the role of membrane
lesions in the cytotoxic action of 6-TG, we have determined
the effects of 6-TG on fucose metabolism in a susceptible
transplanted neoplasm. The findings demonstrate that ex
posure of Sarcoma 180 cells to 6-TG for as little as 1 hr in
vivo results in an inhibition of [3H]fucose but not
[14C]glucosamine incorporation into cell glycoproteins.
Such an inhibition of the incorporation of a terminal carbo
hydrate may be involved in the modification of surface
architecture caused by this drug. The inhibition by 6-TG of
fucose utilization for glycoprotein formation appears to be
due largely to a decreased activation of fucose to GDPfucose in drug-treated cells.
MATERIALS
AND METHODS
6-TG was generously provided by Dr. George H. Hitchings
of the Burroughs Wellcome Research Laboratories (Re
search Triangle Park, N. C.). [14C]Glucosamme, [3H]glucosamine, [3H]fucose, and GDP-['"C]fucose were pur
chased from New England Nuclear, (Boston, Mass.).
Fischer's medium and horse serum were obtained from
Grand Island Biological Co., (Grand Island, N. Y.).
Sarcoma 180 ascites cells were collected from the ascitic
fluids of female CD-1 mice (Charles River Breeding Labora
tories, North Wilmington, Mass.) 6 to 8 days after i.p.
inoculation of mice with 6 x 106 tumor cells. In studies of
the in vivo effects of 6-TG, animals were given injections of
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6-7G Effects on Fucose Incorporation
5 to 8.8 /jCi (0.07 to 0.12 ^g) of [3H]fucose and/or 2.5 /¿Ci the use of a column (2.5 x 17 cm) of Sephadex G-25
(9.5 /¿g)of [14C]glucosamine per mouse, and 30 min were (Pharmacia, Piscataway, N. J.) and 50 mw ammonium
acetate as eluent. The effluent containing GDP-fucose was
allowed for incorporation unless otherwise stated. Contam
of 0.1 M TCA,
inating erythrocytes were removed by washing cells 3 times lyophilized to dryness, resuspended in 200 ¿J
with 10 to 15 volumes of a Ca2'-Mg2 -free phosphate- and heated to 90°for 10 min. After 4 extractions with 10
buffered NaCI solution (8.0 g NaCI, 0.2 g KCI, 2.16 g
NaJ-tPO,-7H2O, and 0.2 g KH,PO, per liter of H,0, pH 7.4)
following centrifugation at 75 x g for 5 min at room
temperature. For in vitro experiments, washed cells (4 x
106 cells/ml) were incubated at 37°in the absence or
presence of 6-TG in Fischer's medium supplemented with
5 mM potassium phosphate (pH 7.0) and 10% horse serum.
At various times thereafter, [3H]glucosamine or [3H]fucose
(0.5 /uCi/ml) was added, and the incorporation of radioac
tivity into total cellular glycoprotein was measured by addi
tion of 10 ml of ice-cold 10% TCA to 0.25 ml of either
untreated or treated cells. Pellets were collected by centrif
ugation at 4°and washed consecutively with 10 ml of icecold 5% TCA (twice), 5 ml of chloroform:methanol:ether
(2:2:1. v/v), and 5 ml of methanol. The pellet was hydrolyzed
with 1 ml of 1 M NaOH at 80°for 30 min and then neutralized
with 5 M HCI. Radioactivity was determined with a Packard
Tri-Carb liquid scintillation spectrometer (Packard Instru
ments Co., Downers Grove, III.). The incorporation of radio
activity after exposure of cells to 6-TG and radioactive
tracer in vivo was determined in a similar manner. Washed
cells were resuspended in 10 volumes of Ca2'-Mg2 -free
phosphate-buffered NaCI solution (1.64 x 107 cells/ml),
and 1-ml samples were added to 10 ml of ice-cold 10%
TCA. The resulting precipitate was collected by centrifuga
tion and washed twice with 10 ml of ice-cold 5% TCA. Less
than 15 and 27% of the acid-precipitable radioactive fucose
and glucosamine products, respectively, were removable
with chloroform:methanol:ether
(2:2:1, v/v). The pellet was
hydrolyzed and neutralized, and its radioactivity was deter
mined as described above.
The intracellular concentrations
of [3H]fucose and
GDP-[3H]fucose were measured by the method of Yurchenco and Atkinson (27). Briefly, washed radiolabeled
cells were extracted twice with 5 to 6 volumes of 60%
ethanol in a boiling water bath for 5 min each. The com
bined extracts were centrifuged at 1,000 x g for 5 min, and
the supernatant solution was recentrifuged at 33,000 x g
for 20 min. The supernatant fraction was evaporated to
dryness under vacuum at 30°,redissolved in 1 ml of water,
and centrifuged at 33,000 x g for 20 min to remove ethanolsoluble but water-insoluble material. Descending Whatman
No. 3MM paper chromatography was carried out with 95%
ethanol:1.0 M ammonium acetate (7:3, v/v) for 9 hr. Airdried paper strips were cut into 1-cm pieces and extracted
twice with 60% ethanol in a boiling water bath. The extracts,
which contained 90% of the radioactivity, were evaporated
to dryness, and radioactivity therein was determined in
Aquasol (New England Nuclear). The counting efficiency in
all single-labeled experiments ranged between 15 and 30%
for 3H and 31 and 35% for MC and radioactivity for doublelabeled experiments was 17% for 3H and 27% for I4C, with
40% 14Cto 3H spillover.
In those experiments in which the specific activity of the
GDP-fucose was determined, GDP-fucose was separated
from other ethanol-soluble, water-insoluble materials with
volumes of ether, the solution was chromatographed on
Whatman No. 3MM paper for 6 hr with n-butyl
alcohol:pyridine:water (6:4:3, v/v) as solvent (5). After sep
aration, L-fucose was eluted from the paper, and its concen
tration was measured according to the method of Diseñe
and Shettles (6).
RESULTS
The rate of [3H]fucose incorporation into glycoprotein
was measured following exposure of Sarcoma 180 cells to
60 MM 6-TG for either 2 or 6 hr (Chart 1). A linear rate of
fucose incorporation into acid-precipitable material oc
curred for up to 1 hr in untreated Sarcoma 180 cells that
had been preincubated for 2 or 6 hr. 6-TG markedly de
creased the rate of utilization of [3H]fucose for cellular
glycoprotein synthesis under both experimental conditions.
The rate of [3H]glucosamine incorporation into the glyco
protein of both untreated and 6-TG-treated Sarcoma 180
cells under similar conditions is shown in Chart 2. Un
treated Sarcoma 180 cells incorporated radioactive gluco
samine into acid-insoluble material at essentially a linear
rate for at least 1 hr. At both concentrations of 6-TG (6 and
60 /¿M),an enhanced incorporation of [3H]glucosamine
was observed after the 2-hr preincubation, but it decreased
after 6 hr of drug exposure. However, this reduction in
[3H]glucosamine utilization observed after 6 hr of exposure
to 60 /UM 6-TG was significantly less than that seen with
[3H]fucose.
For more detailed examination of the differential effects
of 6-TG on the incorporation of these precursors into
glycoproteins, cells were incubated with a range of concen
trations of 6-TG (0.6 to 100 ¡¿M)
for 2, 4, or 6 hr (Chart 3).
B
30
60
90
120
30
60
90
120
TIME OF INCUBATION (min)
Chart 1. Incorporation of [3Hjfucose into glycoproteins of Sarcoma 180
cells treated with 6-TG in vitro. Sarcoma 180 cells isolated from ascites fluid
were preexposed to 0.9% NaCI solution or 6-TG (60 MM)for 2 hr (A) or 6 hr
(B). After drug treatment, [3H]fucose (0.5 ¿iCi/ml)was added, and cells were
incubated at 37°for various periods of time. Radioactivity present in TCAprecipitable material from 10* cells was determined. Each value represents
the mean of results obtained from duplicate samples of 2 separate experi
ments. •
, control; G, 6-TG.
DECEMBER 1977
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4251
J. S. Lazo et al.
the time of exposure to the 6-purinethione was increased
to 4 or 6 hr, the incorporation of glucosamine into acidinsoluble material was gradually decreased as a function of
both concentration and time, with a maximum inhibition of
35% occurring after 6 hr of exposure to 100 /J.M6-TG.
The in vivo incorporation of [3H]fucose into acid-precipitable material of Sarcoma 180 ascites cells was essentially
linear for at least 30 min after the i.p. injection of 0.07 ¿¿g
of fucose per mouse (Chart 4A). Similarly, [14C]glucosamine
B
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-i
LJ
O
m
8
Z
o.
o
20
40
60
20
40
60
TIME OF INCUBATION (min)
Chart 2. Incorporation of [3H]glucosamine into glycoproteins of Sarcoma
180 cells treated with 6-TG in vitro. Sarcoma 180 cells, preexposed to 0.9%
NaCI solution or 6-TG (6 or 60 /¿M)
for 2 hr (A) or 6 hr (B), were incubated
with [3H]glucosamine (0.5 Ã-Ã-Ci/ml)
at 37°for various periods of time. Radio
activity present in TCA-precipitable material from 106cells was determined.
Each value represents the mean of results obtained from 2 to 3 separate
experiments. •,control; D, 6-TG, 6 /¿M;
A, 6-TG, 60 ¡J.M.
2hr
120
incorporation into macromolecules was linear for 30 min
(data not shown). Cells from mice treated with 6-TG (20
mg/kg) 2 hr before exposure to 6-TG had significantly less
radioactivity in both acid-soluble and acid-precipitable frac
tions at 30 and 60 min (Chart 4). The reduction in [3H]fucose
incorporation into glycoprotein was time dependent; it
occurred as early as 1 hr after exposure to 6-TG and
reached a maximum of 65% inhibition at 2 hr (Chart 5).
Inhibition of [3H]fucose incorporation by 6-TG persisted for
up to 6 hr after the administration of the purine antimetab
olite. By 24 hr after the drug, however, the utilization of
fucose for glycoprotein synthesis was enhanced. No signif
icant decrease in the rate of [14C]glucosamine incorporation
into glycoprotein was detected; although, in a manner
similar to [3H]fucose, [14C]glucosamine utilization for the
formation of glycoproteins was increased at 24 hr. In both
the acid-precipitable and acid-soluble fractions, the inhibi
tion after a 2-hr exposure of Sarcoma 180 to 6-TG reached
100
4hr
O
¡E 80
Z
o
u
o
6hr
60
40
o
IO
O
*v
Z
CL
io-
io"
io-1
-4
IO
O
[e-TGMOLARITY]
Chart 3. Effects of various concentrations of 6-TG on [3H]fucose and
[3H]glucosamine incorporation into glycoproteins of Sarcoma 180 cells in
vitro. Sarcoma 180 cells were exposed to various concentrations of 6-TG
(0.6 to 100 /J.M)for either 2, 4, or 6 hr. [3H]Glucosamine (0.5 ¿iCi/ml)or
[3H]fucose (0.5 /iCi/ml) was added after 6-TG treatment and incubated for 1
hr. Determination of the incorporation of fucose and glucosamine into
glycoproteins was performed as described in Chart 1. Each value represents
the mean of results obtained from 2 separate experiments. [3H]Fucose
incorporation: O, 2 hr; D, 4 hr; A, 6 hr. [3H]Glucosamine incorporation; •,
2 hr; •4 hr; A. 6 hr.
Fifty % inhibition of [3H]fucose utilization for the synthesis
of glycoproteins occurred at 8, 5, and 4 /¿M6-TG after
respectively, 2, 4, and 6 hr, of incubation with this agent.
The incorporation of [3H]glucosamine into glycoprotein
was enhanced at all concentrations of 6-TG tested after 2
hr of preincubation with the purine antimetabolite but, as
4252
IS
30
60
MINUTES AFTER [3h]
FUCOSE
Chart 4. The effect of 6-TG on the incorporation of [3H]fucose into
Sarcoma 180 cells in vivo. Sarcoma 180-bearing mice were given i.p.
injections of 0.9% NaCI solution or 6-TG (20 mg/kg; 0.3 to 0.4 ml) 2 hr prior
to injection of 5 fiCi [3H]fucose per mouse. At various times thereafter, cells
were collected and washed 3 times. The radioactivity associated with the
TCA-insoluble (A) and TCA-soluble (B) material was calculated as the
average of separate determinations from 2 to 3 mice. Bars, S.E. or range of
values; •,0.9% NaCI solution; D, 6-TG.
CANCER
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6-TG Effects on Fucose Incorporation
a maximum at a 10-mg/kg dose of drug (Chart 6). Control
cells incorporated 25 pg of [3H]fucose per 106 cells into
glycoproteins after injection of 8.8 /¿Ci/permouse, and
cells from animals treated with 10 mg 6-TG per kg incorpo
rated only 4.5 pg of [3H]fucose per 106 cells into these
macromolecules. The [3H]fucose utilization was measured
in a subline of Sarcoma 180 resistant to 6-TG (S180/TG), in
which the mechanism of resistance involves an increased
catabolism of the active nucleotide form(s) of the antimetabolite by elevated levels of alkaline phosphatase (28).
Table 1 shows that the decrease in [3H]fucose incorporation
into glycoproteins of Sarcoma 180 by 6-TG did not occur to
the same degree in S180/TG cells. Thus, a 77% reduction
in the rate of [3H]fucose utilization for glycoproteins of
Sarcoma 180 cells was observed 2 hr after 6-TG (10 mg/
kg), whereas only a 38% reduction was detected in S180/
TG cells under the same conditions. Similarly, a 45%
decrease in acid-soluble radioactivity from [3H]fucose was
produced by the antimetabolite in Sarcoma 180 cells, while
no decrease was caused by this treatment in S180/TG
cells. These data support the concept that the inhibition of
glycoprotein synthesis by 6-TG requires 6-TG nucleotide
and may be part of the mechanism of cytotoxicity of this
drug.
Since Nelson ef al. (20) have shown that GTP levels are
decreased in neoplastic cells treated with 6-TG, it was
important to determine whether the mechanism by which
6-TG reduced [3H]fucose incorporation into glycoprotein
was due to an inability to activate fucose to the level of
30
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B
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50
60
25
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10
40
4
6
24
HOURS AFTER 6-TG
Chart 5. Incorporation of [3H]fucose and [14C]glucosamine into Sarcoma
180 cells in vivo at various times after 6-TG. Sarcoma 180-bearing mice were
given i.p. injections of 0.9% NaCI solution or 6-TG (20 mg/kg; 0.3 to 0.4 ml).
At various times thereafter, either 5 /¿Ci [3H]fucose or 7.5 ¿iCi
("Cjglucosamine were injected ¡.p.into each mouse. Cells were collected
30 min later, and radioactivity in the TCA-precipitable material was measured
as previously described. Points, average ±S.E. of separate determinations
from 6 to 9 mice. The range of control ["Cjglucosamine incorporation (•)
was 3.6 to 6.3 dpm/103 cells. The range of control [3H]fucose incorporation
(O) was 2.4 to 3.7 dpm/103 cells.
5
6-TG
IO
15
DOSE (mg/kg)
20
Chart 6. The effect of various dose levels of 6-TG on [3H]fucose incorpo
ration into acid-soluble and acid-insoluble material of Sarcoma 180 cells.
Sarcoma 180-bearing mice were given i.p. injections of various doses of 6TG 2 hr prior to being given injections of 8.8 (¿Ci
of [3H]fucose. Radioactivity
was determined as described in Chart 1. Points, average of triplicate
determinations from a single mouse. A, acid-precipitable radioactivity; B.
acid-soluble radioactivity.
Table 1
The effects of 6-TG on [3H]fucose incorporation into glycoproteins of Sarcoma 180 and S 180/TG
cells
Mice were given i.p. injections of 0.9% NaCI solutions with or without 6-TG (10 mg/kg) and 2 hr
later were given i.p. injections of [3H]fucose (5 ¿¿Ci/mouse).Cells were collected 30 min later, and
radioactivity
present in acid-precipitable
material was determined. Triplicate determinations
were
made of samples from each animal.
pg ¡3H]fucose equivalents incorpo
pg [3H]fucose equivalents incorpo
rated/106 Sarcoma 180/TG cells"
rated/106 Sarcoma 180 cells"
Treatment0.9%
NaCI solution
6-TGAcid
insoluble10.7
±1.7(7)"
soluble47.0
±2.7 (7)
21.0 ±0.6 (4)Acid
2.4 ±0.4 (4)Acid
" Mean ±S.E.
* Numbers in parentheses, number of mice per group.
DECEMBER
insoluble13.7
soluble43.5
±1.6 (7)
±10.4 (5)
8.5 ±0.9 (7)Acid 40.8 ± 3.4 (6)
1977
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4253
J. S. Lazo et al.
GDP-fucose. For this reason the intracellular content of
[3H]fucose and GDP-[3H]fucose was measured by the
method of Yurchenco and Atkinson (27). The ethanol-soluble radioactivity extracted from whole cells was identical
with that found in TCA-soluble fractions (49.8 ±9.3 versus
47.0 ±2.7 pg of [3H]fucose equivalents per 106cells). GDP[3H]fucose represented 93.5% of the radioactivity found in
control cell extracts (Chart 7). The only other 3H-labeled
species extracted from cells was [3H]fucose, which repre
sented 4.9% of the recovered radioactivity. Cells obtained
from mice treated with 20 mg of 6-TG per kg contained
only 9.1 pg of [3H]fucose equivalents per 106 cells in the
ethanolic extracts, while untreated cells had 49.8 pg of
[3H]fucose equivalents per 106 cells (Table 2). Only 75.4%
of the radioactivity recovered from 6-TG-treated cells was
found to be present in GDP-fucose, while over 20.6% of the
radioactivity from 6-TG-treated cells migrated as free fucose. The specific activity of the GDP-fucose pool was
measured in cells from mice treated with 0.9% NaCI solution
or with an identical solution containing 6-TG (20 mg/kg).
Table 3
Specific activity and intracellular
content of GDP-fucose in
Sarcoma 180 cells labeled with [3H]fucose after treatment with 6TG (20 mg/kg)
The specific activity of the GDP-fucose pool was measured in 2
experiments in which cells from 2 mice were exposed in vivo for 2
hr to 0.9% NaCI solution with or without 6-TG (20 mg/kg). The
intracellular
content of GDP-fucose was calculated from the spe
cific activity of GDP-fucose and the total radioactive content of
this nucleotide sugar per cell as reported in Table 2.
GDPfucose per 107
Treatment0.9%
NaCI solution
6-TGnmoles
cpm//j.g
cells2660
GDP-fucose
± 70"
2650 ±810
13.6
2.0
" Mean ±range.
No change in the specific activity of the GDP-fucose pool
was detected, but an 85% decrease in the total intracellular
quantity of GDP-fucose was observed following treatment
with 6-TG (TableS).
DISCUSSION
Q.
Ü
S£2 -
IO
I5
MIGRATION
20
25
30
(cm)
Chart 7. Chromatographie tracing of ethanol extracts from Sarcoma 180
cells after treatment with 6-TG. Sarcoma 180-bearing mice were given i.p.
injections of 0.9% NaCI solution or 6-TG (20 mg/kg) 2 hr prior to being
given injections of 5 /¿Ci
of [3H]fucose. Cells were collected 30 min later and
washed 3 times. Radiolabeled ethanol extracts from •,0.9% NaCI solution
and D, 6-TG-treated cells were subjected to Whatman No. 3MM paper
chromatography with 95% ethanoM M ammonium acetate (7:1, v/v) as
solvent for 9 hr. Arrows, origin and solvent fronts. [3H]Fucose and GDP[14C]fucose migrated with RKvalues of 0.76 and 0.29, respectively.
[3H]Fucose
Sarcoma
scribed in
migrated in
[14C]fucose
determination
Table 2
incorporation
into ethanol extracts of Sarcoma 180
after treatment with 6-TG (20 mg/kg)
180-bearing
mice were treated and assayed as de
Chart 7. The radioactivity
found in the peaks that
a manner identical with that of [3H]fucose and GDPused as standards was calculated from the separate
of cells from 3 to 4 mice. The [3H]fucose used in
these experiments
had 47.2 cpm/pg.
Total pg
[3H]fucose
Treatment
0.9% NaCI solution
6-TG
" Mean ±S.E.
4254
equivalents/
106 cells"
49.8 ±9.3°
9.1 ±3.9
% total radioactivity
GDP-[3H]fucose
93.5 ±1.6
75.4 ±8.7
| 'H]fucose
4.9 ±1.0
20.6 ±6.7
Radioactive fucose is a terminal carbohydrate of glycoproteins and has been extensively used to study the biosyn
thesis of these molecules in eukaryotic cells (e.g., see
Refs. 1, 3, and 15). L-Fucose is metabolically converted to
/3-L-fucose-1-phosphate which is subsequently further anabolized by GDP-L-pyrophosphorylase to form GDP-L-fucose
(14); GDP-fucose is then added to appropriate glycoprotein
and glycolipid acceptors (3). In HeLa cells the radioactivity
incorporated from fucose appears almost exclusively in
plasma membranes in the form of fucosyl glycoproteins
(1). However, the rate at which the radioactive fucose is
incorporated into glycoprotein is not necessarily a reflec
tion of the true rate of synthesis of these proteins, since
the soluble precursor pool, primarily GDP-fucose, can be
derived from 2 sources, i.e., through exogenous fucose
(14) and endogenously synthesized GDP-mannose (8). In
this investigation, after tumor-bearing mice were given
injections of 0.07 /¿gof [3H]fucose, glycoprotein and the
soluble precursor pool (almost completely GDP-fucose)
contained radioactivity equivalent to 10.7 and 47.0 pg of
[3H]fucose per 106 cells, respectively (Table 1). Since the
intracellular pool size of GDP-fucose in Sarcoma 180 was
found to be on the order of 13.6 nmoles (8.3 ¿¿g)/107
cells
(Table 3), it is evident that only trace amounts of [3H]fucose
are being incorporated into glycoproteins under the condi
tions used. Sarcoma 180 cells, like HeLa cells (27), incor
porate [3H]fucose almost exclusively into glycoprotein and
contain predominantly GDP-fucose as the soluble radiolabeled fucose intermediate after a pulse of [3H]fucose
(Chart 7; Table 2).
Exposure of Sarcoma 180 cells to 6-TG both in vitro and
in vivo caused an inhibition of [3H]fucose incorporation
into glycoprotein without markedly reducing [14C]glucosamine incorporation into these macromolecules. This prefer
ential decrease in exogenous fucose utilization occurs
within 1 hr of drug treatment (Chart 4) and appears to be
due, at least in part, to a decrease in intracellular GDPfucose content (Table 3). It is well established that 6-TG
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6-7G Effects on Fucose Incorporation
and the related antimetabolite 6-mercaptopurine inhibit
the de novo biosynthesis of purine nucleotides (22, 23) and
thereby cause a decrease in the intracellular pool of GTP
(20). Such inhibition would be expected to lead to the
suppression of purine nucleotide sugar biosynthesis by
reducing the contents of both GDP-fucose and GDPmannose. The observed decrease in the content of GDPfucose in 6-TG-treated cells is consistent with such a
concept. Moreover, since endogenously synthesized GDPmannose appears to be the major source of GDP-fucose
(14), the failure to see a decrease in GDP-fucose specific
activity after 6-TG treatment (Table 3) suggests that GDPmannose formation is also inhibited by the purine antime
tabolite. Previous results from our laboratory indicate that
at 6 hr, but not at 2 hr, after 6-TG treatment, a significant
decrease occurred in the rate of cellular agglutination
produced by the mannose-specific lectin, concanavalin A
(11,12). This depression in the rate of agglutination appears
to be due to a decrease in the degree of binding of
concanavalin A to Sarcoma 180 cells (J. S. Lazo and A. C.
Sartorelli, unpublished data). It has been demonstrated
that surface components are associated with tumorigenicity, cell proliferation, and DNA replication (16, 21, 25).
Moreover, it appears that at least some neoplastic tissues
have significantly higher fucosyltransferase and fucosidase
activities and higher GDP-fucose content than does normal
tissue (2). Thus, it is conceivable that the alteration or
inhibition of carbohydrate addition to surface membranes
of tumor cells may disrupt the biosynthesis of cell surface
receptors for external factors (e.g., serum factors) neces
sary for cell proliferation (10). This suggestion is supported
by the fact that inhibition of exogenous fucose incorpora
tion is 1 of the earliest events inhibited by 6-TG and is sus
tained for several hr after exposure of cells to this agent.
Several other possible consequences of 6-TG inhibition of
glycoprotein biosynthesis include osmotic imbalance and
altered membrane transport, since these processes depend
on membrane glycoprotein (13). Our results indicate that
6-TG has effects on surface glycoprotein biosynthesis and
suggest that this phenomenon may be of relevance to the
cytotoxicity produced by this agent.
ACKNOWLEDGMENTS
We wish to thank Charles W. Shansky for his technical assistance.
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Downloaded from cancerres.aacrjournals.org on April 20, 2017. © 1977 American Association for Cancer Research.
4255
Inhibition of l-Fucose Incorporation into Glycoprotein of
Sarcoma 180 Ascites Cells by 6-Thioguanine
John Stephen Lazo, Kou M. Hwang and Alan C. Sartorelli
Cancer Res 1977;37:4250-4255.
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