[CANCER RESEARCH 39, 3677-3682, September 1979]
0008-5472/79/0039-0000
$02.00
Correlation of Glycosylation in a Membrane Protein with a Molecular Weight
of I 50,000 with Tumorigenic Property of Rat Fibrosarcoma Variant&
Kotaro Koyama,2 Edward Nudelman, Minoru Fukuda, and Sen-itiroh Hakomori3
Division of Biochemical Oncology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98 104, and Departments of Microbiology and Pathobiology,
University of Washington, Seattle, Washington 98195
ABSTRACT
Four clonal variants of fibrosarcoma cells with differing de
gnees of tumonigenicity and intercellular adhesive property
have been isolated from a methylcholanthnene-induced tumor
produced in inbred Donryu rats. The variant clones G and Z
were characterized by an extremely low tumorigenicity in con
trast to the high tumonigenicity of the original clone A and the
variant clone P. The variant P was highly tumonigenic in the
ascites form when inoculated in the penitoneal cavity, but its
tumonigenicity was minimal when inoculated s.c. With scanning
electron micrognaphy, sharp well-developed microvilli were
observed on the surface of clones Z and G in contrast to
smooth blebs on the surface of clones A and P. Clone G cells
showed a remarkable side by side adherence to form a worm
like shape, sharing a common cell coat. Clone Z showed a
similar tendency but to a lessendegree. Clones A and P did not
show adherent property.
Protein and glycoprotein profiles of these variant cell lines
have been studied bythe following surface-labeling techniques:
lactoperoxidase-catalyzed [1251]iodination; galactose oxidase
NaB3H4; sialidase-galactose oxidase-NaB3H4; peniodate
NaB3H4; and by metabolic labeling with precursor sugars.
These variant cell lines show a remarkable difference in degree
of sialylation in one glycoprotein species with a relative molec
ular weight of 150,000, whereas the profiles of other proteins
and glycoproteins are indistinguishable between variant cell
lines. The possible role of glycosylation in the ectoprotein
(M.W., 150,000) in definingthe tumonigenicpropertyof variant
cell lines is discussed.
INTRODUCTION
Phenotypes of tumor cells are variable. Some mutant cell
lines induced by mutagens show a low tumonigenicity and are
called ‘
‘
revertants' ‘
(12, i 3, 23, 24), whereas some variants
selected by repeated passage in animals show a high tumori
genicity (4, •
17). The high-tumonigenic phenotype of various
continuous tumor cell lines can be suppressed by cell fusion
with low-tumonigenic L-cells, whereby one chromosome seems
to be gained. Therefore, the tumonigenic phenotype may result
from the loss of a growth control gene in one chromosome
which is supplemented by nontumorigenic cells through cell
1 This
study
is supported
by United
States-Japan
Cooperative
Program
for
Cancer Research and by Research Grant CA23907 from the National Cancer
Institute.
2 Recipient
of
the
Exchange
Research
Grant,
United
States-Japan
Cooperative
Program for Cancer Research. Present address: National Cancer Center Re
search Institute, Tsukiji 5-chome, Chuo-ku, Tokyo, Japan.
3 To
whom
requests
for
reprints
should
be
addressed,
at
Fred
Hutchinson
Cancer Research Center, 1124 Columbia Street, Seattle, Wash. 98104.
Received April 16, 1979: accepted June 13, 1979.
SEPTEMBER 1979
fusion (27, 28). A number of hepatocarcinoma variants (Yosh
ida hepatoma) with different growth characteristics have been
investigated, and their degree of malignancy has been corre
lated with their glycolytic isozyme pattern. The process of
phenotypic reversion from malignant to nonmalignant status
was termed ‘
‘decarcinogenesis'
‘
(13, 25). The mutant clones
showing decarcinogenesis were derived from the ascites form
of mammary carcinoma by mutagens (18).
Tumonigenic phenotypes of cells have been correlated with
the profile of cell surface membrane molecules. The following
molecular changes of membranes have been most extensively
studied and have been regarded as the common phenotype for
some tumors: incomplete synthesis of glycolipids and associ
ated accumulation of their precursors (2, 10, i 1); the appear
ance of a sialosyl glycopeptide unique to the transformed cells
(9, 26); and the loss of a high-molecular-weight glycoprotein
called fibronectin or Gap a (8, 14). A glycoprotein, with a
molecular weight of 100,000, was detected in a wide range of
tumonigenic cells which showed less binding ability to wheat
germ lectin than to Con A4 as compared to a similar glycopro
tein of nontumonigenic cells (3). However, relatively few studies
have been made on membrane changes as related to pheno
typic revertant on variant cells derived from decarcinogenesis.
Four phenotypic variants with a remarkable difference in
tumorigenicity were isolated from a methylcholanthrene-in
duced tumor of inbred Donryu rats by K. Koyama at the
National Cancer Center Institute (Tokyo). Subsequently, their
cell surface proteins and glycoproteins have been analyzed at
the Fred Hutchinson Cancer Research Center and the Univer
sity of Washington, Seattle, Wash. This paper reports a note
worthy finding, namely, that the status of glycosylation in a
unique glycoprotein with a molecular weight of 150,000 may
reflect the tumonigenic properties of 4 variant isolates.
MATERIALS AND METHODS
History and Establishment of 4 Clones. The original tumor
was a spindle-cell sarcoma induced by s.c. injection of 0. 1%
of methylcholanthrene in inbred Donryu rats. Tumor cells were
released by trypsinization of minced sarcoma and inoculated
at about I 0@cells into the peritoneal cavity of Donnyu rats.
Finally, the original free-cell type ascites sarcoma AS 653 was
established. A highly metastatic variant was isolated from the
original AS 653 cells by s.c. inoculation of sarcoma cells
followed by trypsinization of metastatic lymph nodes. This was
repeated i 0 times; thus, the highly metastatic clone (termed
as AS 653-HM) was isolated. The AS 653-HM was further
selected by colony formation in 0.3% soft agar in Eagle's
medium supplemented with 10% calf serum. Cloning was re
4 The
abbreviation
used
is:
Con
A,
concanavalin
A.
3677
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K. Koyama et al.
peated 3 times, thus establishing the highly malignant clone A.
Clone G was isolated from clone A by culturing in Eagle's
medium containing i % calf serum for more than i 30 genera
tions. During this term, some cells become confluent to form
an unusual worm-like appearance (see Fig. 1G). It appears that
the cell surface coats of these cells are fused, and cells are
linked together through a common surface coat. These linked
cells proliferate as a linked state. The worm-like confluent cells
were isolated mechanically and continuously cultured in the
medium containing 1% calf serum. The morphology and growth
behavior of clone G were not altered as long as it was cultured
in a low concentration of calf serum.
Clone P was isolated from clone A by culturing in a soft agar
plate with Eagle's medium containing 10% calf serum.
Clone Z was isolated from clone A by its adhesive property
on glass surfaces. Clone Z adhered on glass whereas clone P
did not.
The growth rate in vitro of the various clones was examined
in Eagle's medium containing 10% fetal calf serum. Transplant
ability was tested by inoculation of i O@cells s.c. and i.p., and
the percentage of survivors was determined by days.
Cell Culture. Cells were grown as a suspension in Dul
becco's modified Eagle's medium supplemented with various
concentrations of calf serum or fetal calf serum as specified in
the presence of kanamycin (i 00 @zgIml).
Metabolic Labeling of Cells. Cells were cultured in the same
medium described above and supplemented with 10% fetal
calf serum, but containing [3H]glucosamine (2 @CiImlmedium)
for 48 hr. After incubation, cells were collected by centnifuga
tion, washed with phosphate-buffered saline (20 mp@i
sodium
phosphate buffer, pH 7.0, containing 0.1 5 M NaCI), and ana
lyzed.
Cell Surface Profile of Proteinsand Glycoproteins.Proteins
and glycoproteins of these cells were analyzed by polyacryl
amide slab gel electrophoresis in sodium dodecyl sulfate ac
cording to the method of Laemmli (19). The total protein profile
was examined using Coomassie blue staining (5). Cells were
collected by centnifugation (800 x g), and a homogeneous cell
suspension was prepared by mechanical agitation. Cells (2 x
10@) suspended in about i ml of phosphate-buffered saline
were subjected to various surface-labeling procedures. The
surface-exposed proteins were labeled by lactoperoxidase-cat
alyzed 1251iodination (22). Cell surface glycoproteins were
labeled by 3 methods: galactose oxidase and NaB3H4;sialidase
and galactose oxidase treatment followed by reduction with
NaB3H4(7), and sodium metapeniodate treatment followed by
reduction with NaB3H4for sialic acid labeling (6). The incuba
tion time for labeling cells was short (i to 3 hr), and cells
appeared to be viable after labeling. Glycoproteins were Ia
beled by these methods, separated by polyacrylamide gel
electrophoresis, and visualized by fluorognaphy (i).
seen in Chart i . In this experiment, 10@cells were inoculated
i.p. in the inbred Donryu rats. Clone P did not grow following
s.c. inoculation, although its malignancy in ascites form was
greater than the original clone A. The cell surface structure of
these clones as revealed by scanning electron microscopy is
shown in Fig. 1. It is noteworthy that clone G which showed a
remarkably low tumonigenicity (Chart 1) was characterized by
an unusual confluent figure with a worm-like shape; i.e. , several
cells are confluent to form a chain. Microvilli appear sharp and
needle-like. Clone Z cells which also showed low tumorigenicity
were characterized by their sharp microvilli and tendency to
attach together. Transplantability, chromosome mode, and his
tocompatible antigenicity of these clonal variants are summa
nized in Table 1.
Cell Surface Glycoproteinsof 4 Variant Clones. The protein
profiles of the 4 variant cell lines stained by Coomassie blue
showed very similar patterns (see Fig. 2), and the surface
labeling patterns of proteins by lactoperoxidase-catalyzed 12@1
iodination were indistinguishable among the 4 variant cell lines
(data not shown). Although we have not examined the protein
pattern separated by 2-dimensional gel electrophoresis, the
protein components of these 4 variant cell lines may not be
significantly different.
In contrast to the protein label, the labeling of galactosyl or
N-acetylgalactosaminyl residues by galactose oxidase and tnt
iated sodium borohydnide showed a remarkable difference.
Both clone Z and clone G hardly gave any label in contrast to
a striking label at the molecular weight i 50,000 region of
clones P and A (see Fig. 3 left). However, intensive label
occurred at a molecular weight of 150,000 and at other bands
in clones G and Z after sialidase and galactose oxidase treat
ment. After sialidase treatment, however, the molecular weight
150,000 band of clone P showed only moderately intensified
label, and that of clone A did not show greatly intensified label
(see Fig. 3 right). Thus, sialylgalactosyl or sialyl-N-acetylgalac
tosaminyl residues in molecular weight 150,000 glycoproteins
are much greater in number in G and Z clones than in variants
P and A. The results of direct sialosyl labeling with peniodate
NaB3H4 indicated that the sialosyl residue in the molecular
weight i 50,000 glycoprotein is much greater in variants G and
Z than in variants P and A (Fig. 4). The pattern of glycoprotein
metabolically labeled with [3H]glucosamine did not show a clear
distinction between clones G, Z, and P, A, since the label is
incorporated heavily in various protein species including cyto
plasmic proteins (Fig. 5).
w
a:
-J
RESULTS
In Vitro and in Vivo Growth Characteristics of 4 Variant
Clones. The growth curves of 4 variants showed essentially
the same growth rate in Eagle's minimum essential medium
supplemented with 10% fetal calf serum (data not shown).
However, malignancy, as measured by the ability of these cells
to grow progressively in vivo, showed a striking difference as
3678
>
U)
DAYS
Chart 1. Survival rate of Donryu rat after inoculation of ascites forms of
methylcholanthrene-induced fibrosarcoma and their variants. Ascites, original AS
653 cells as in the text: A, clone AS 653-HM: P Z, and G, variants as explained
in the text. Cells (10@)were inoculated on Day 0 and observed until Day 55. Each
group consists of 10 rats. Mean values of 3 experiments are shown.
CANCERRESEARCHVOL. 39
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150K Glycoprotein
of Rat Sarcoma
Variants
Table 1
variantsTransplantabilitySolidtumorAscitesHostChromo
Biological and morphological properties of rat fibrosarcoma A5-653
abilityaFormMicro
no.AntigenAS-653HM++9-10Single±67R*1
mationformatlonkilling
Clonesfor
villibsome
(D)cA++18Single±53R*i(D)P—+25Single+49RW1
(D)Z+—50Slightly
(D)G+—50Linked
452R*i
linked+
(D)
together++37-65R*1
@
a The mean survival days after 1o@cells were inoculated i.p.
b , •
÷, mean
degree
ofmicrovilli
development
(seeFig.1).
C Rat
histocompatibility
antigen
R,
Identical
to
those
found
in
Wistar
strain
(R'@),
Group
1 (R*1
)
In
addition,
all
tumor
cells
contained
a
“non
R' ‘
antigen specific for the Donryu strain which is assigned as (D). These antigens were determined by inhibition of rat hemagglutination (16).
DISCUSSION
The results of this study clearly indicate that Iow-tumonigenic,
low-invasive variants G and Z were characterized by a higher
degree of labeling for sialosyl galactose or sialosyl-N-acetyl
galactosamine groups in a specific glycoprotein with a relative
molecular weight of 150,000. In striking contrast, the same
glycoprotein of the highly tumonigenic original clone A showed
a much lower degree of labeling for the same structures. The
clone P, which showed preferential growth as an ascites form
but did not grow as a solid tumor, was also characterized by a
lesser degree of labeling. It is noteworthy that the profiles of a
number of other proteins and glycoproteins of these variant
cell lines were indistinguishable. It was noticed that the less
tumonigenic G and Z clones have better developed microvilli
than highly tumonigenic clones A or P. A positive correlation
between the surface-labeling profile and the cell surface mor
phology is interesting but requires further extensive study.
Recently, Bramwell and Harris (3) found that a membrane
protein having a relative molecular weight of 100,000 in a wide
range of tumorigenic cells showed affinity to wheat germ lectin
and Con A different from that of a similar glycoprotein of
nontumonigenic cells. The glycoprotein derived from tumoni
genic cells showed less affinity to wheat germ lectin than to
Con A, whereas the glycoprotein from nontumonigenic cells
showed an opposite affinity to the 2 lectins. This indicates that
the glycoprotein of less tumorigenic cells may contain a higher
proportion of carbohydrate ‘
‘peripheralstructure,' ‘
such as
sialic acid and N-acetylglucosamine which bind to wheat germ
lectin, than of the core mannose structure which binds to Con
A. The results of the present study also clearly indicate that
the glycoprotein of less tumonigenic cells contained a larger
quantity of the peripheral structure consisting of sialosyl-galac
tose or N-acetylgalactosamine than that of highly tumonigenic
cells. In our previous studies, less tumonigenic variants as
compared to highly tumonigenic hybrids were characterized by
the presence of more complex carbohydrate chains in glycolip
ids and an increased quantity of a certain ganglioside (15).
It is obvious, therefore, that some carbohydrate chains linked
either to a protein or to a lipid showed a great deal of simplifi
cation in tumonigenic cells. However, glycopeptides derived
from a number of transformed cells by extensive proteolysis
consistently showed a ‘
‘higher
molecular weight' ‘
than those
derived from nontransformed cells (9, 26). This difference of
SEPTEMBER 1979
glycopeptide profile demonstrated on gel filtration was inter
preted as a core structural difference by Ogata et a!. (21 ). Our
present finding and those of Bramwell and Harris (3) could be
an additional cell surface parameter that indicates tumorigenic
ity. Recently, Yogeeswaran et al. (29) described the surface
exposed glycolipid and glycoprotein of in vivo selected mela
noma cell lines which showed different metastatic properties.
In their study, however, highly metastatic cells showed a
greater concentration of sialosylgalactosyl or sialosyl-N-ace
tylgalactosaminyl label which is different from our present
results. More recently, Lloyd et al. (20) demonstrated the
presence of a common surface-labeled glycoprotein in various
clones of human melanoma cells.
All of these studies strongly support the idea that a certain
change of glycosylation pattern in a defined membrane protein
may occur during transformation and that the pattern of gly
cosylation may define cell social behavior. As a consequence,
this change may influence the degree of tumonigenic and
invasive properties of tumor cells. The nature of abnormal
glycosylation has been clearly defined in glycolipids (10, 11);
however, that occurring in glycoproteins has not been well
defined. It may be a matter of abnormal processing in glyco
sylation, and thus may cause an anomalous branching struc
ture as has been postulated by Ogata et al. (21 ). Abnormal
sialylation patterns of the different clones indicated in this study
may also be related to the change in ‘
‘core
structure' ‘
of the
glycoprotein.
ACKNOWLEDGMENT
The authors are grateful to Dr. Takashi Sugimura, National Cancer Center
Research Institute, Tokyo, Japan for his kind arrangement of this study through
the U.S-Japan
Cooperative Program for Cancer Research.
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CANCERRESEARCHVOL. 39
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150K Glycoprotein of Rat Sarcoma Variants
p
A
z
1
G
Fig. 1. Scanning electron mlcrographs of variant clones of rat fibrosarcoma. Clones A, P, Z, and 0 are indicated by each letter. Magnification of the pictures of
clones A, P. Z, and G, x 5,800, x 6,400, x 7,800, and x 4,600, respectIvely.
SEPTEMBER
1979
368i
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@
@—
@
4
@.
K. Koyama et al.
2
@
.‘,.,,,‘
0@' ..
3
@:r-@@•- @
-
@
@-.-
-
_
--
@—-@
G
-
PZA
GPZA
GPZA
I
*@
@.
P1
f@
GPZA
GPZA
@
4
Fig. 2. Total protein bands of various rat fibrosarcoma clones stained by Coomassie blue. Lanes G, P, Z, and A, clones G, P, Z, and A, respectively. Note that total
protein pattern of each clone is identical and that high-molecular-weight components are hardly detectable. Arrow, protein (MW., 150,000): the molecular weight
marker used for this determination is the same as in Fig. 3.
Fig. 3. Cell surface glycoprotein pattern of various clones of rat fibrosarcoma. Cells were labeled with galactose oxidase-NaB3H4and shown on the left or labeled
with sialidase-galactose-oxidase-NaB3H4and shown on the right. Lanes G, P. Z, and A, are clones G, P. Z and A, respectively. Each gel contained 125 @zg
of protein
(left) or 100
of protein (right). Note that the original clone A showed an intense band at molecular weight of 150,000 without neuraminidase treatment (A, left)
(arrow). The band was not intensified after neuraminidase treatment (A, right). The same band was not stained by galactose oxidase alone for clone G and clone Z
(G, left:
@
Z, left).
These
bands
became
strongly
stained
after
neuraminidase
treatment
(G, right:
Z, right).
The staining
behavior
of the same
band
for clone
P was
intermediate, namely, the band appeared with galactose oxidase alone but significantly intensified after neuraminidase treatment. The following standard proteins
were used for estimation of apparent molecular weight in polyacrylamide gel electrophoresis: skeletal myosin, 200,000: Escherichia coIl RNA polymerase (8' and
subunit), 165,000 and 155.000: bovine serum albumin, 68,000: ovalbumin, 43,000.
Fig. 4. The glycoprotein pattern labeled by periodate-Na3HB4of various rat fibrosarcoma clones. Lanes G, P. Z, and A, clones G, P, Z, and the original clone A,
respectively. Note that the band for molecular weight of 150,000 (arrow) labeled strongly for clones G and Z, but not for clones P and A. This finding supports the
presence of a highly sialylated carbohydrate chain at molecular weight of 150,000 protein in clones G and Z, supporting the results as shown in Fig. 3.
Fig. 5. Metabolic labeling of various glycoproteins of rat fibrosarcoma clones. Lanes G, P, Z, and A, clones G, P, Z, and A, respectively. Note that the pattern of
total carbohydrate labeled by glucosamine is entirely different from those indicated by cell surface labeling. Arrow, Protein (MW., 150,000).
3682
CANCERRESEARCHVOL. 39
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Correlation of Glycosylation in a Membrane Protein with a
Molecular Weight of 150,000 with Tumorigenic Property of Rat
Fibrosarcoma Variants
Kotaro Koyama, Edward Nudelman, Minoru Fukuda, et al.
Cancer Res 1979;39:3677-3682.
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