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[CANCER RESEARCH 35, 63-67, January 1975]
Autoradiographic Localization of Glycoprotein in Human Breast
Cancer Cells Maintained in Organ Culture after Incubation with
[3H]Fucose or [3H]Glucosamine'
Gerald B. Dermer and Russell P. Sherwin
Department ofPathology, Hospital of the Good Samaritan Medical Center, Los Angeles, California 9tXfl7 [G. B. D.], and Department of Pathology,
University ofSouthern California School ofMedicine, Los Angeles, California 9t'X@33
[R. P. S.]
SUMMARY
Explants
of nine infiltrating
duct
carcinomas
of the
human female breast, maintained in organ culture, were
exposed to the glycoprotein precursors, L-[3H]fucose and
[3H]glucosamine, in order to determine the cellular distribu
tion of newly synthesized glycoprotein
as revealed by
autoradiography
with the light and electron microscopes.
Explants were incubated with a single isotope for 24 hr, at
which time some of the labeled explants were removed for
autoradiographic
analysis while the rest were transferred to
nonradioactive medium for an additional 24 hr.
After exposure to label for 24 hr. autoradiography
with
each isotope was similar and showed strong reactions over
most tumor cells. The reactions were due to clumps of silver
grains over intracytoplasmic
lumina within single tumor
cells and silver grains over Golgi saccules, cytoplasmic
vesicles, lysosome-like
bodies, lateral and basal plasma
membranes,
and microvilli. Extracellular
ductular struc
tures were also heavily labeled. At the later sampling time,
Golgi saccules often showed a reduced reaction while the
reactions over other organdies and intracellular and extra
cellular ductular structures remained strong.
The observations suggest that in our in vitro system the
tumor cells are metabolically
active and complete the
synthesis of the carbohydrate
side chains of glycoproteins
within the Golgi apparatus. From there, some of the newly
synthesized glycoprotein
appears to migrate to plasma
membranes
and lysosome-like bodies. Furthermore,
our
data support the notion that many duct carcinomas of the
breast exhibit secretory activity by showing that some newly
synthesized glycoprotein also appears to become products
that are secreted into intracellular and extracellular ductu
lar structures.
INTRODUCTION
Within a cell, glycoproteins
are found primarily as
components of plasma membranes (4), lysosomes (8),'and
1 Supported
in
part
by
Contract
NOl-CB-23860
within
Cancer Task Force of the National Cancer Institute.
Received July 19, 1974; accepted September 23, 1974.
JANUARY
the
Breast
secretory material (22). Fucose is a terminal residue (17) of
some of the oligosaccharide side chains of glycoproteins and
is incorporated
directly (5, 12) into newly synthesized
glycoprotein within saccules of the Golgi apparatus (2, 3).
Glucosamine is also efficiently incorporated into glycopro
tein (10, 11), but in this case some glucosamine is converted
to other hexosamines or sialic acid before incorporation into
the growing oligosaccharide chains (10). Since these hexosa
mine residues are located nearer the polypeptide chain than
terminal fucose or sialic acid residues are, they are incorpo
rated earlier into newly synthesized glycoprotein.
This
occurs within the rough endoplasmic
reticulum as the
polypeptide migrates through the endoplasmic reticulum to
the Golgi apparatus.
Autoradiographic
studies ( 1-3) of
many normal cell types have shown that, after completion
of the sugar side chains in the Golgi apparatus, glycoprotein
migrates to cell surfaces, to lysosomes, and into secretory
products. The intensity of the autoradiographic
reaction
over organdies depends on the cell type (3).
Here we report our experiments designed to find out the
cellular distribution
of newly synthesized
glycoprotein
within human breast carcinoma cells maintained for several
days in organ culture.
MATERIALS
AND METHODS
The specimens used in this study consisted of small pieces
of human female breast tumors obtained under sterile
conditions minutes after mastectomy. The data presented
here are confined to observations from 9 infiltrating duct
carcinomas.
The tumor pieces were sliced under sterile
conditions into 1-mm cubes and explanted on triangular
stainless steel grids within organ culture dishes (Falcon
Plastics Co., Oxnard, Calif.) containing 1 ml of medium and
placed in an incubator at 37°. The culture medium (16)
consisted of Eagle's double-strength
amino acid medium
supplemented with 20% calf serum and 5% beef embryo
extract ultrafiltrate.
Also incorporated
were glucose, 500
mg/ 100 ml; penicillin, 100 units/mI; and streptomycin, 100
@og/ml.After culture for 24 hr. 100 @.tCiof L-[6-3H]fucose
(New England Nuclear, Boston, Mass.; specific activity,
13.4 Ci/mmole) or 100 @sCiof D-[6-3Hjglucosamine
hydro
chloride (New England Nuclear; specific activity, 10.13
Ci/mmole) were added to different dishes each of which
1975
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63
G. B. Dermer and R. P. Sherwin
contained 4 to 6 explants. Incubation with the labeled
compounds proceeded for 24 hr, at which time several
explants from each dish were removed for autoradiographic
analysis at the light and electron microscopic level. The
remaining
explants
were transferred
to nonradioactive
me
dium for an additional 24 hr and then also processed for
subsequent autoradiographic analysis.
Correlation of data from the light and electron microscope
made it clear that the discrete, strong reactions over the
cytoplasm of many tumor cells observed with the light
microscope corresponded to the heavy labeling of intracyto
piasmic lumina observed in the electron microscope.
Smooth-surfaced
tracytoplasmic
vesicles in the cytoplasm around in
lumina also exhibited
an autoradiographic
reaction (Fig. 3). At the later sampling time, the vesicies
for I to 2 hr in a 2% solution of glutaraldehyde in 0.2 M near lumina often lacked a reaction while the reaction
The initial steps in processing involved fixing the explants
sodium cacodylate buffer (pH 7.3), postfixing for 2 hr in 1%
osmium tetroxide in cacodylate
buffer, dehydrating
in
within lumina remained
strong (Fig. 4).
Electron microscopy also confirmed our tentative light
microscope observations regarding ductuiar structures by
acetone, and embedding in the polyester resin Vestopal W.
For light microscope autoradiography,
1.0-@sm-thick showing that the frequently encountered
unstained
sections of the Vestopal-embedded
explants were
coated with Kodak NTB 2 emulsion (Eastman Kodak Co.,
Rochester, N. Y.) by dipping the slides on which the
ductular
characteristically
had strong autoradiographic
The small lumens of many ductules were bounded
cells exhibiting microvilli (Figs. 5 and 6), and
structures
reactions.
by several
the silver
sections had been placed into melted emulsion diluted 1:1
with distilled water (6). The slides were stored at 5°in
grains were frequently found over lumens (Figs. 5 and 6),
lightproof boxes for I week and then developed. After
near the luminal surfaces (Fig. 6).
Electron microscope autoradiography also revealed that
the general cytoplasmic reaction seen over tumor cells at
both sampling times with the light microscope was actually
localized for the most part over Golgi saccules and associ
ated smooth-surfaced vesicles (Fig. 7), lysosome-like bodies
autoradiography,
the sections
emulsion with toluidine blue.
were stained
through
the
For electron microscope autoradiography, thin (silver to
gold) sections from the same explants were autoradio
graphed with Ilford L4 emulsion (Ilford Ltd., Ilford, Essex,
England) by the loop method of Maunsbach (14). Sections
microvilli
(Figs.
5 and 6), and smooth-surface
vesicies
of carbon. By means of the loop, a monolayer film of
(Fig. 8), and lateral and basal plasma membranes (Fig. 9).
A small percentage of the grains also appeared over
cisternae of the rough endoplasmic reticulum. At the later
sampling time, the Golgi reaction was often decreased.
emulsion
Other organelles within the tumor cells exhibited an insig
placed on Formvar-coated
grids were stained with uranyl
acetate and lead citrate and then covered with a thick coat
was then placed over individual
grids. Emulsion
melted at 42°was used diluted I: 1 with distilled water. The
grids were then stored in lightproof
nificant autoradiographic
reaction.
boxes at 5°for about 1
month and then developed for 90 sec in undiluted Kodak
Microdoi-X and fixed for 4 mm in 30% sodium thiosulfate.
DISCUSSION
The strong autoradiographic
RESULTS
Light microscope examination
of the explants showed
that most tumor cells exhibited a strong autoradiographic
reaction
over the cytoplasm
after incubation
with
F3H]fucose
or
[3Hjglucosamine.
The
pattern
of
reaction
was
similar with each isotope. In addition to a general cytoplas
mic reaction, discrete clumps of silver grains were found
over what appeared to be intracytoplasmic
lumina (Fig. 1).
Few grains were found over nuclei. Sometimes the distribu
tion of label appeared to change with time; at the later
sampling time (24 hr incubation with label followed by 24 hr
in nonradioactive
medium), the general autoradiographic
reaction over tumor cell cytoplasm was often decreased
while the reactions over lumens of ductular structures (Fig.
2) and structures suggestive of intracytoplasmic
lumina
remained intense.
Electron microscopic examination of the explants con
firmed our tentative light microscope observations regard
ing intracytoplasmic lumina, since a striking localization of
grains to intracytoplasmic
lumina (Figs. 3 and 4) within
single tumor cells was indeed present. This reaction was
primarily over the microvilli that bounded these duct-like
spaces and over granular
material within the lumina.
64
reactions we have observed
over the cytoplasm of tumor cells within explants after
incubation with [3H]fucose or [3H]glucosamine
indicate
that, in our organ culture system, the cells are first of all
metabolically
active. Furthermore,
the cells take up and
incorporate sugars into glycoproteins since free fucose, its
nucleotide sugar derivative, and glycolipids are for the most
part extracted from tissue during histological processing,
while glycoproteins are retained (3, 20). Although some free
glucosamine,
as opposed to fucose, may be retained by
tissue (20), the similarity of the autoradiographic
patterns
observed with both isotopes indicates that the reaction
following [3Hjglucosamine addition is again due primarily
to labeled glycoproteins.
The most striking observation was the finding of intense
autoradiographic
reactions over intracytoplasmic
lumina
within single tumor cells. These structures, rarely found in
normal human breast epithelium (7, 9, 13, 19), have been
considered to represent a departure from normal ductular
formation and are believed to contain secretions (7). Our
data are in accord with a secretory activity and suggest that
malignant human breast epithelial cells synthesize glyco
proteins, perhaps similar to I or more components of milk,
which are secreted into intracytoplasmic
lumina. The strong
reactions over microvilli at surfaces of intracytopiasmic
CANCER
RESEARCH
VOL. 35
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Localization
lumina could be largely due to recently released secretory
products.
Moreover,
the findings also imply that the
cytopiasmic vesicies ( I -3) around lumina may be carriers of
secretion products. Secretion is also suggested by the strong
autoradiographic
reactions found over extraceilular ductu
lar structures, reminiscent of normal cells active in glyco
protein secretion (3, 20). However, other explanations to
account for the localization of grains over intracellular and
extraceilular ductular structures are nevertheless possible.
The strong autoradiographic reaction over Golgi saccules
of breast carcinoma cells after 24 hr exposure to isotope
suggests that, as in normal cells (I —3),the Golgi apparatus
may be the primary site of completion of the oligosaccha
ride side chains of glycoprotein. The observation that the
reaction over Goigi zones decreases after labeled explants
are cultured for an additional 24 hr in nonradioactive
medium supports this view and indicates that, as in normal
cells, newly synthesized glycoprotein
migrates from the
Golgi apparatus to other cellular sites and into secretory
products.
Newly synthesized
glycoprotein
also appears to be
incorporated into plasma membranes of malignant human
breast epithelium. Since we do not know the growth rate of
the tumor cells in vitro, we cannot say whether the plasma
membrane reaction was due primarily to a turnover of
glycoprotein components of plasma membranes as observed
in nongrowing cells or to an increase in substance of plasma
membranes as seen in growing cells (18). Strong autoradio
graphic reactions over lysosome-like
bodies within the
tumor cells suggest that, as in normal cells (3), newly
synthesized glycoprotein [presumably hydrolyases (8)] mi
grates to this organelle.
Although different techniques were used, some observa
of Glycoprotein
in Breast Cancer Cells
3. Bennett, G., Leblond, C. P., and Haddad, A. Migration of Glycopro
tein from the Golgi Apparatus to the Surface of Various Cell Types as
Shown by Radioautographyafter LabeledFucoseInjection into Rats.
J. Cell Biol., 60: 258-284, 1974.
4. Bretscher, M. S. Membrane Structure: Some General Principles.
Science,181:622-629, 1973.
5. Coffey, J. W., Miller, N., and Sellinger, 0. Z. The Metabolism
L-Fucose in the Rat. J. Biol. Chem., 239: 401 I -4017, 1964.
of
6. Dermer, G. B. An Autoradiographic and BiochemicalStudy of Oleic
Acid Absorption by Intestinal Slices Including Determinations of
Lipid Loss during Preparation for Electron Microscopy. J. Ultra
struct. Res., 22: 312-325, 1968.
7. Goldenberg, V. E., Goldenberg, N. S., and Sommers, S. C. Compara
tive Ultrastructure of Atypical Ductal Hyperplasia, Intraductal Carci
noma and Infiltrating Ductal Carcinoma of the Breast. Cancer, 24:
1152-1169,1969.
8. Goldstone, A., and Koenig, H. Lysosomal Hydrolases as Glycopro
tein. Life Sci., 9: 1341-1350, 1970.
9. Haguenau,F. Significanceof Ultrastructure in Virus-InducedTumors.
NatI. Cancer Inst. Monograph, 4: 21 I -249, 1960.
10. Hughes, R. C., Sanford, B., and Jeanloz, R. W. Regeneration of the
Surface Glycoproteins of a Transplantable Mouse Tumor Cell after
Treatment with Neuraminidase. Proc. NatI. Acad. Sci. U. S., 69:
942-945, 1972.
I I. Kapeller, M., Gal-Oz, R., Grover, N. B., and Doljanski, F. Natural
Shedding of Carbohydrate-Containing Macromolecules from Cell
Surfaces.Exptl. Cell Res., 79: 152-158, 1973.
12. Kaufman, R. L., and Ginsburg, V. The Metabolism
of i-Fucose by
HeLa Cells. Exptl. Cell Res.,50: 127-132, 1968.
13. Kern, W. H., and Dermer, G. B. The Cytopathology of Hyperplastic
and Neoplastic Mammary Duct Epithelium. Acta Cytol., 16: 120-129,
1972.
14. Maunsbach,A. B. Absorption of P25-LabeledHomologous Albumin
by Rat Kidney Proximal Tubule Cells. A Study of Microperfused
Single Proximal Tubules by Electron Microscopic Autoradiography
and Histochemistry. J. Ultrastruct. Res., 15: 197-241, 1966.
tions of cell cultures of mouse mammary tumors seem to
parallel our observations regarding the secretory activity of
human breast carcinomas. The mouse cell cultures exhibited
“domes,―
which appeared to be periodically expanding and
collapsing
secretory
structures
(2 1). Furthermore,
the
domes contained milk constituents (15) that seemed to be
produced by the cells participating in the dome structure.
IS. McGrath, C. M., Nandi, S., and Young, L. Relationship between
Organization of Mammary Tumors and the Ability of Tumor Cells to
Replicate Mammary Tumor Virus and to Recognize Growth Inhibi
ACKNOWLEDGMENTS
18. Warren, L., and Glick, M. C. Membranes of Animal Cells. II. The
Metabolism and Turnover of the Surface Membrane. J. Cell Biol., 37:
729-746, 1968.
tory Contact Signals In Vitro. J. Virol., 9: 367-376, 1972.
16. Sherwin, R. P., Richters, V., Brooks, M., and Buckley, R. D. The
Phenomenonof MacrophageCongregationIn Vitro and Its Relation
ship to In Vivo NO, Exposure of Guinea Pigs. Lab. Invest., 18:
269-277,1968.
17. Spiro, R. Glycoproteins: Their Biochemistry, Biology and Role in
Human Disease.New EngI. J. Med., 281: 991-1001, 1969.
The authors wish to acknowledge the excellent technical assistance of
Carole Kaufman and Valda Richters.
19. Wellings, S. R., and Roberts, P. Electron Microscopy of Sclerosing
Adenosis and Infiltrating
Duct Carcinoma of the Human Mammary
Gland. J. NatI. Cancer Inst., 30: 269-287, 1963.
REFERENCES
1. Bennett, G. Migration of Glycoprotein from Golgi Apparatus to Cell
Coat in the Columnar Cells of the Duodenal Epithelium. J. Cell Biol.,
45. 668-673, 1970.
2. Bennett, G., and Leblond, C. P. Formation of Cell Coat Material for
the Whole Surface of Columnar Cells in the Rat Small Intestine as
Visualized by Radioautography. J. Cell Biol., 49: 409-416, 1970.
20. Whur, P., Herscovics, A., and Leblond, C. P. Radioautographic
Visualization of the Incorporation of Galactose-3H and Mannose-3H
by Rat Thyroids In Vitro in Relation to the Stages of Thyroglobulin
Synthesis. J. Cell Biol., 43: 289-31 1, 1969.
21. Visser, A. S., and Prop, F. J. A. “Domes,―
Periodically Explanding
and Collapsing Secretory Structures in Cell Cultures of Mouse
Mammary Tumors. J. NatI. Cancer Inst. 52: 293-294, 1974.
22. Winterburn, P. J., and Phelps, C. F. The Significance of Glycosylated
Proteins. Nature, 236: 147-151, 1972.
JANUARY 1975
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65
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The autoradiographsare from explantsof infiltrating duct carcinomasof the human breastthat weremaintained in organ culture and incubatedeither
with i-['H]fucose
or [‘H]glucosamine. Sections examined in the light microscope (Figs. I and 2) were stained with toluidine blue while the electron
autoradiographs(Figs. 3 to 9) were from sectionsstainedwith uranyl acetateand lead citrate.
Fig. I . Explant incubatedwith [°Hjfucosefor 24 hr. Besidesa generalautoradiographic reaction over the cytoplasm of tumor cells, clumps of silver
grains are found over intracytoplasmic
lumina (arrows). Original magnification,
x 1,000.
Fig. 2. Explant incubatedwith [3H]glucosaminefor 24 hr and then transferredto nonradioactivemedium for an additional 24 hr. Comparedto Fig. I,
the cytoplasmic autoradiographic reaction is decreasedwhile many silver grains are still found over the lumen of a ductular structure. Original
magnification, x 1,000.
Fig. 3. Explant incubated with [3H]glucosamine for 24 hr. An intense autoradiographic reaction is found over an intracytoplasmic
single tumor cell. Cytoplasmic vesicles around the lumina also exhibit a reaction. x I I ,000.
66
CANCER
lumina within a
RESEARCH
VOL. 35
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research.
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of Glycoprotein
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Fig. 4. Explant incubated with [3Hjglucosamine for 24 hr and then transferred to nonradioactive medium for an additional 24 hr. Similar to Fig. 3, but
vesicles around the intracytoplasmic lumina show few silver grains. x I 1,000.
Fig. 5. Explant incubated with [3H]glucosamine for 24 hr and then transferred to nonradioactive medium for an additional 24 hr. The small
extracellular lumen bounded by several tumor cells exhibits a strong autoradiographic reaction. x I 1,000.
Fig. 6. Explant incubated with [3Hlglucosamine for 24 hr. Similar to Fig. 5, but cytoplasmic vesicles near luminal surfaces also exhibit an
autoradiographic reaction . x 16,500.
Fig. 7. Explant incubated with [3H]fucose for 24 hr. Many silver grains are found over Golgi saccules and associated smooth-surfaced vesicles. x
16,500.
Fig. 8. Explant incubated with [3H]glucosamine for 24 hr and then transferred to nonradioactive medium for an additional 24 hr. Lysosome-like bodies
within a tumor cell exhibit an autoradiographic reaction. x I 1,000.
Fig. 9. Explant incubated with [3H]fucose for 24 hr. Silver grains are found over lateral plasma membranes. x I 1,000.
JANUARY
1975
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67
Autoradiographic Localization of Glycoprotein in Human Breast
Cancer Cells Maintained in Organ Culture after Incubation with [
3H]Fucose or [3H]Glucosamine
Gerald B. Dermer and Russell P. Sherwin
Cancer Res 1975;35:63-67.
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