[CANCER RESEARCH 32, 380-389, February 1972]
Effects of D-Glucosamine, D-M annosa mine, and 2-Deoxy-Dglucose on the Ultrastructure of Ascites Tumor Cells in Vitrol
Z. Molnar and J. G. Bekesi
Department of Pathology, University of Chicago, Chicago, Illinois 60637 [Z. M.J, and Department of Medicine A, Roswell Park Memorial
Institute, 666 Elm Street, Buffalo, New York 14203 ¡J.G. B.]
SUMMARY
Ehrlich ascites carcinoma and Sarcoma 180 ascites tumor
cells were treated in vitro with amino sugars, and the resulting
changes were studied by light and electron microscopy.
Addition of D-glucosamine or D-mannosamine
to the
incubation medium provoked striking cytoplasmic and nuclear
changes. The earliest changes, seen after incubation for 15
min, included vacuolization of the cytoplasm and separation
of the electron-lucent filamentous parts of the nucleolonema
and nucleolar vacuole at the periphery of the nucleolus. As the
time of incubation increased, vacuolization of the cytoplasm
increased gradually, accompanied by retraction
of the
cytoplasm around the nucleus. After complete extrusion of its
electron-lucent components, the nucleolus became condensed.
At the end of 3 hr of incubation, 95% of the tumor cells had
pycnotic, polymorphic, or disintegrating nuclei, and the
nucleoli in nearly all the cells examined consisted almost
exclusively of the compacted,
granular, electron-dense
nucleolonema. After 4 hr, most tumor cells exhibited various
degrees of disintegration. Incubation of these tumor cell lines
for 4 hr with 2-deoxyglucose resulted in no significant
structural alteration in the ascites tumor cells.
INTRODUCTION
It was reported in a previous paper (2) that D-glucosamine
and D-mannosamine
caused Ehrlich ascites carcinoma,
Sarcoma 37, and Sarcoma 180 ascites cells to lose their
viability and transplantability. Cells exposed to 2-deoxy-D-glu
cose and to most other sugars showed little loss of viability,
and such cells produced tumors when inoculated into mice.
These observations made it desirable to investigate the
ultrastructural changes provoked by D-glucosamine, D-manno
samine, and 2-deoxy-D-glucose in these ascites cells.
Quastel and Cantero (15) administered glucosamine to
tumor-bearing
mice and observed shrinkage of nuclei,
retraction of cytoplasm, and marked eosinophilia in the
neoplastic tissue within 2 hr. Rubin et al. (19) reported that
incubation of Sarcoma 37 cells with D-glucosamine caused
extensive degeneration.
Fjelde et al. (6) observed that
glucosamine caused marked inhibition of the growth of human
'This study was supported in part by Grant P-401 from the
American Cancer Society, and by USPHS Grants CA-05183 and
GRS-FR-5367.
Received September 28, 1971; accepted November 4, 1971.
380
epidermoid carcinoma cells in tissue culture. Histological
examination
showed pronounced
nuclear changes and
granulation of the cytoplasm in the glucosamine-treated
neoplastic cells.
In this investigation, Ehrlich ascites carcinoma and Sarcoma
180 ascites tumor
cells exposed
to D-glucosamine,
D-mannosamine, and 2-deoxy-D-glucose were studied by
means of light and electron microscopy.
MATERIALS AND METHODS
Freshly harvested 7- to 10-day-old Ehrlich ascites carcinoma
and Sarcoma 180 ascites cells were utilized. To 7 ml of
Krebs-Ringer phosphate buffer, pH 7.4, containing 50 unióles
of glucose in a 25-ml Erlenmeyer flask, were added 2 ml of
Krebs-Ringer solution alone or containing 0.5 mmole of
D-glucosamine, D-mannosamine, or 2-deoxy-D-glucose per ml.
After equilibration at 37° for 2 min, l ml of freshly
harvested ascites fluid containing 1.2 to 1.5 X IO8 Ehrlich
ascites carcinoma or Sarcoma 180 ascites tumor cells was
added to each flask. The cells were incubated for 4 hr at 37°in
a Dubnoff incubator equilibrated with air. In a 2nd
experiment with D-glucosamine, Ehrlich ascites carcinoma
cells were incubated for 15, 30, or 45 min or 1,2, 3, or 4 hr.
At the end of incubation, the cells were collected by
centrifugation at 500 X g for 3 to 5 min in a SORVALL
refrigerated centrifuge and then were washed twice with
ice-cold Krebs-Ringer phosphate buffer. Incubated control
cells of both tumor lines were treated in the same manner
except that the test compounds were omitted from the
incubation medium.
For electron microscopy, the washed ascites cells, including
pellets of unincubated control cells, were fixed at 20°for 24
hr in ice-cold 4% paraformaldehyde buffered at pH 7.4 with
s-collidine and containing 0.005 M calcium chloride (13). The
specimens were then cut into 1-mm cubes and postfixed for 45
min in 2% osmium tetroxide buffered at pH 7.4 with
s-collidine. After fixation, the tissues were dehydrated and
then embedded in Epon 812 resin. For light microscopy,
sections approximately 1 ju thick were cut from Epon blocks
and stained with Mallory's azure 2-methylene blue. Ultrathin
sections were cut with diamond knives on a Sorvall MT-2 or
Reichert OM U2 ultramicrotome. Sections were stained with
lead citrate (18) and uranyl acetate (29). Photographs were
made with Kodak electron image plates in a Philips EM 200
electron microscope.
CANCER RESEARCH VOL. 32
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Effects of Amino Sugars on Ascites Tumor Cells
Table 1
Effects of period of incubation with D-glucosamine on the morphology of Ehrlich osciles carcinoma cells
Cells were studied under the light microscope. Epon sections were stained with Mallory's azure 2-methylene blue. Each test
group contained 500 cells.
Nucleus (%)
Group
shape,
Crescent shaped, polyCytoplasmic vacuoles 1 M
of
GlucosamineNoNoYesYesYesPeriod
incubationOhr4hr15 round,(%)706040264C152234447315182330239°16799595
or oval
morphic, or pycnotic
Indistinct
or more in diameter
min(30
min)b1
or 45
hr(2hr)4hr(3
hi)Usual
a The great majority of the vacuoles were small, only a few being larger than l Min diameter.
b Cells incubated for times shown in parentheses did not significantly differ in appearance from cells incubated for times
not in parentheses.
c Although the nuclei had the usual shape, the nuclear matrix underwent a change in appearance, being either more
condensed than normal or else swollen, and the outline of the nuclear membranes was indistinct.
RESULTS
Light Microscopy
Table 1 lists data concerning gross features of the nuclei and
cytoplasm of Ehrlich ascites tumor cells following incubation
with D-glucosamine for various periods of time. Unincubated
Ehrlich ascites cells (Fig. 1) and Sarcoma 180 ascites tumor
cells were indistinguishable in Epon sections stained with
Mallory's azure 2-methylene blue. Tumor cells constituted 90
to 94% of the cell population, the other 6 to 10% cells being
mainly macrophages, polymorphic leukocytes, and mast cells.
The tumor cell population of both ascites tumors consisted
mostly of lightly stained cells and a few dark cells. The nuclei
were round or oval, and the outline of the nuclear membrane
was distinct in a great majority of the cells. Small cytoplasmic
vacuoles less than 1 n in diameter were present in many cells,
but coarser vacuolization was rare. Incubation up to 4 hr with
glucose (Fig. 2) or 2-deoxyglucose resulted in a slight increase
in vacuolization of the cytoplasm without apparent gross
change in the nuclei.
Addition of D-glucosamine resulted in pronounced changes.
Even incubation for only 15 min caused coarse vacuolization
in 79% of the Ehrlich ascites cells. The degree of involvement
of individual cells varied, ranging from single large vacuoles to
numerous large vesicles often occupying half of the cytoplasm
of a cell. The nuclei of less than half of the cell population
retained their usual structure; the others were crescent shaped,
irregular, or indistinct. Incubation for 30 or 45 min caused
essentially no further changes.
Incubation
for 1 hr resulted in pronounced
coarse
vacuolization of the cytoplasm of 95% of the cells (Fig. 3),
and continued incubation for 2, 3, or 4 hr caused no further
gross change in the cytoplasm. In contrast, although the nuclei
still showed their usual appearance after 1 or 2 hr, incubation
for 3 or 4 hr resulted in loss of the usual outline of the nucleus
in 96% of the cells (Fig. 4). Not even the remaining 4% of
round or oval nuclei qualified as retaining their usual structure
because their nuclear matrices appeared to have changed and
FEBRUARY
their nuclear membranes were indistinct. Ehrlich ascites
carcinoma cells and Sarcoma 180 ascites cells incubated for 4
hr in the presence of D-mannosamine were indistinguishable
from the same types of cells treated with D-glucosamine.
Electron Microscopy
Fine Structure of Unincubated Ehrlich Ascites Carcinoma
and Sarcoma 180 Cells. The morphology of untreated Ehrlich
ascites tumor cells in this study was similar to that reported by
others (5, 22, 30, 31). Some features are described here,
however, because they were especially noteworthy or because
they played an important role in this study. The tumor cell
population consisted mostly of electron-lucent cells and a few
somewhat smaller electron-dense cells, with a spectrum of cells
ranging between the 2 types.
The electron-lucent cells had large, oval nuclei containing 1
or 2 irregularly rounded nucleoli (Fig. 5). The zones of the
nucleolus were usually rather indistinct because the meshwork
of the nucleolus was mostly tightly woven around small,
electron-lucent parts. Nucleolar vacuoles (26) were present in
almost all nucleoli (Figs. 5 and 6), occasionally communicating
with the nuclear matrix through what appeared to be a narrow
opening (Fig. 6).
The nuclear matrix contained scattered, small clumps of
interchromatin particles. In the cytoplasm of these cells, the
organelles were loosely arranged. The matrices of the larger
vesiculated mitochondria and the cisternae of the RER2
contained a loosely arranged, finely filamentous material. The
membranes of the cisternae of the RER were, in the majority
of the cells, 600 to 1000 A apart. Occasionally, short segments
of the RER were vesicular and were up to 0.9 ¡j.
wide. Most of
these widened parts of the RER contained numerous viral
particles of the A type (1, 7, 23). Fine, long, cytoplasmic
filaments about 70 A in diameter were most abundant in the
perinuclear area, where they appeared to be associated with
2The abbreviations used are: RER, rough endoplasmic reticulum;
RNP, ribonucleoprotein.
1972
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381
Z. Mainar and J. G. Bekesi
the outer membrane of the nuclear envelope. They enmeshed
cytoplasmic organdÃ-es, particularly in the Golgi zone (Fig. 8).
At higher magnifications, these filaments appeared beaded,
with a 30- to 40-Â substructure.
Membrane-limited
bodies
(lysosomes?)
with
a
heterogeneous
content
(10) contained
aggregated RNP
particles, markedly electron-dense amorphous material, and
seemingly unassembled components of viral particles. Virus or
virus-like particles were never observed within the nucleus.
C-type virus particles were occasionally
seen in the
extracellular space.
The electron-dense
cells contained
densely packed
cytoplasmic elements with large numbers of RNP particles in
the peripheral cytoplasm (Fig. 6). The nuclei of these
electron-dense cells were usually pleomorphic. They showed
large nucleoli with a nucleolonema more densely compacted
than that in the electron-lucent cells. Highly electron-dense
granules measuring up to 600 A in diameter were frequently
observed in the nucleolar vacuoles of cells of this type (Fig. 6)
and occasionally in the tumor cells of the intermediate
electron
density.
Like the electron-lucent
cells, the
electron-dense cells showed microvilli with fine filamentous
cores on their surfaces.
Sarcoma 180 tumor cells were indistinguishable from
Ehrlich ascites cells (Fig. 7). Like the latter, the cells of this
line contained A-type virus particles in the cisternae of the
RER. What appeared to be unassembled components of virus
particles (capsid material?) were seen within membrane-limited
bodies, and unlike such material in Ehrlich ascites cells, they
were also seen as free aggregates in the cytoplasm. The
particles of those aggregates were ring shaped, of uniform size,
measuring approximately
230 A in diameter, with an
electron-lucent center (Fig. 9).
Fine Structure of Incubated Control Ehrlich Ascites and
Sarcoma 180 Tumor Cells. Incubation up to 4 hr in the
presence of glucose in Krebs-Ringer buffer resulted in no
significant alterations in the fine structure of the tumor cells.
In a few tumor cells, short segments of the cisternae of the
RER were dilated, measuring more than 1 ju in diameter.
Effects of D-Glucosamine and D-Mannosamine on the Fine
Structure of Ehrlich Ascites Carcinoma and Sarcoma 180
Ascites Cells in Vitro. Addition of D-glucosamine or
D-mannosamine
to the incubation medium resulted in
pronounced
morphological
alterations in Ehrlich ascites
carcinoma and Sarcoma 180 ascites cells. After treatment with
D-glucosamine
for 15 min, the nucleoli were tightly
compacted.
The electron-lucent
filamentous components
became isolated at the surface of the nucleolus, forming round
aggregates that were usually opposite the nucleolar vacuole
(Fig. 10). Such separation was not observed in the more than
500 untreated control ascites tumor cells examined with the
electron microscope. In the cytoplasm, globular aggregates of
RNP particles formed. Mitochondria of many cells showed
focal swelling with loss of the fine, filamentous matrix. Large
vesicles walled with smooth membrane were frequently seen in
the Golgi zone.
Incubation for 30 or 45 min, resulted in essentially the same
changes, with a pronounced decrease in the electron density of
the peripheral cytoplasm in most of the cells at 45 min (Fig.
11). The nucleolar vacuoles had an unusually wide surface
382
contact with the nuclear matrix, probably representing a step
in gradual expulsion from the tightening meshwork of the
nucleolonema.
Incubation for 1 or 2 hr resulted in progressive decrease in
the electron density of the isolated filamentous components,
and these aggregates, including the nucleolar vacuole, became
further separated (Figs. 12 and 13). Because of their similar
consistency, it became difficult, if not impossible, to
distinguish between what may have been the nucleolar vacuole
and the previously scattered electron-lucent
filamentous
components. The nuclear matrix became highly condensed and
contained abundant clumps of interchromatin granules. The
cytoplasm was conspicuously vesiculated.
Further incubation for 3 hr resulted in a much-diminished
cytoplasm (Fig. 14). Electron-lucent filamentous components,
including nucleolar vacuoles, were very rarely recognizable.
Incubation of both tumor cell lines with either of the 2
amino sugars for 4 hr resulted in disintegration of the cells
(Figs. 15 and 16). Most cells examined indicated some degree
of necrosis. The nucleolus was coarsely granular in all cells; but
in contrast to the effect of actinomycin D (16, 21, 25), further
separation of the electron-dense filamentous component of the
nucleolonema from the granular part was not observed in this
study. The nuclear matrix appeared to be coagulated or
clumped. In about 20% of more than 200 cells examined with
the electron microscope, the nuclear envelope was focally
disrupted, spilling nuclear chromatin into the adjacent
cytoplasm (Fig. 16). In about 25% of the cells, mitochondria
contained focal, electron-dense granular deposits in the
intercristal space (Figs. 15 and 16), like those in the groups
treated with 2-deoxy-D-glucose (Fig. 17).
Effects of 2-Deoxy-D-glucose on the Fine Structure of
Ehrlich Ascites Carcinoma and Sarcoma 180 Ascites Cells in
Vitro. The most conspicuous change involving both tumor cell
lines at the end of 4 hr of incubation with 2-deoxy-D-glucose
was the swelling of mitochondria and the formation of round
aggregates of RNP particles (Fig. 17). The fine, filamentous
substance within the mitochondrial matrix had a looser
arrangement than in the control groups. Much as in the case of
treatment with sugar amines, approximately 20% of the cells
treated with 2-deoxy-D-glucose contained focal, electron-dense
deposits on short segments of mitochondrial cristae and in the
adjacent intercristal space (Fig. 17). These unidentified
deposits consisted of granules 30 to 50 A in diameter. The
nuclei and nucleoli, however, showed no change.
DISCUSSION
Addition of D-glucosamine or D-mannosamine to the
incubation medium caused severe, irreversible effects on
Ehrlich ascites carcinoma and Sarcoma 180 ascites tumor cells;
vacuolization and retraction of the cytoplasm ¡deformation of
the nucleus, as evidenced by bizarrely shaped, pleomorphic
nuclei; and, most obviously, separation of the nucleolar
electron-lucent components.
Previous viability and transplantability
studies (2) have
indicated that incubation of cells from any of several ascites
tumor lines with D-glucosamine results in death of the
neoplastic cells. The biosynthesis of protein, RNA, and DNA
CANCER RESEARCH VOL. 32
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Effects of Amino Sugars on Ascites Tumor Cells
by neoplastic tissues was strongly inhibited by glucosamine in
vivo and in vitro (3). In addition, the size of the pool of
UDP-TV-acetylhexosamine was markedly increased in cells
exposed to glucosamine (4). At the same time, there was a
significant reduction in the pools of other nucleotides,
particularly UMP, UDP, UTP, and UDPG, in the cancer cells.
Evidence is not yet available to indicate whether any of these
changes are the primary cause of death of the neoplastic cells.
In view of the close relationship of the nucleolar vacuoles to
RNA synthesis in the nucleolus (12), it is significant that the
separation of the electron-lucent components of the nucleolus,
particularly the progressive extrusion of the nucleolar vacuoles
in this study, form the most conspicuous morphological
evidence for the toxicity of the amino sugars. The nucleolar
changes resembled, to some degree, the effect of actinomycin
D (16, 21, 25). In contrast to the situation with amino sugars,
however, lesser nuclear and cytoplasmic changes accompanied
the separation of nucleolar components after treatment with
actinomycin D.
The present results obtained with Ehrlich ascites cells
exposed to D-glucosamine for various periods of time suggest
that complete cellular alterations, both cytoplasmic and
nuclear (including nucleolar), may be necessary before changes
in the tumor cells become irreversible. These results
substantiate data obtained by other methods (2—4, 24)
indicating that glucosamine, galactosamine, and mannosamine
will, under specific conditions, lethally damage ascites tumor
cells.
Focal cytolysis and rarefaction of the peripheral zones of
the cytoplasm, observed in this study in most of the cells at
the end of 1, 2, 3, or 4 hr of incubation with amino sugars, are
interpreted as advanced changes in a necrotizing cell. These
results are similar to those obtained by Goldberg and Green
(8), who studied the cytotoxic properties of antitumor-cell
antisera on Krebs ascites tumor cells. The structural changes
observed in this study were unlike those occurring in Ehrlich
ascites tumor cells after exposure to hypotonie conditions (9)
in which the cell membrane was found to be rather well
preserved even in cells showing pronounced cytoplasmic
alteration.
Previous experiments indicate that exposure of cells of these
tumor cell lines to 2-deoxy-D-glucose causes minimal loss of
viability and that the exposed cells produce tumors when
inoculated into mice (2). Such findings may be explained by
the results of this ultrastructural study which showed that
addition of 2-deoxy-D-glucose to the incubation medium
induced cytoplasmic damage but left nuclei and nucleoli
unchanged.
The electron-dense
material found within
mitochondria
in cells treated with 2-deoxy-D-glucose,
D-glucosamine, or D-mannosamine was not analyzed. It may
represent ferritin taken up from hemolyzed red blood cells. In
view of the similar deposits found in mitochondria from rats
treated with calciferol (14), however, as well as the known
rapid and massive calcium-binding capacity of mitochondria in
vitro (28), it seems most likely that the electron-dense deposits
in this study were amorphous calcium salts.
The
present
electron
microscope
observations
of
unincubated control Ehrlich ascites tumor cells generally
confirm the findings of earlier investigators (5, 22, 30, 31).
The differences in the electron density of tumor cells in this
FEBRUARY
study may represent different stages in the cell cycle or in
functional activity or may be due to differences in the state of
hydration of the cells. There is also a distinct possibility that
the electron-dense cells are not tumor cells, although the large,
atypical nucleoli, the usually abundant virus particles in the
endoplasmic reticulum of these cells, and the presence of cells
of intermediate electron density argue against this possibility.
The cytoplasm of both ascites tumor cell lines contained a
rather dense network of fine filaments around the nucleus and
in the Golgi zone. This finding is somewhat different from
those of Wessel and Bernhard (30) and may be due to the
difference in fixation methods or to differences in the Ehrlich
ascites tumor cell lines examined. Also, the RER in the cells in
this study appears to be more developed than in the cells
reported on earlier (30). Virus particles, present in almost all
of the cells examined, were of the A type reported by
Friedlaender and Moore (7) and by Adams and Prince (I) to
occur in 8 sublines of Ehrlich ascites.
Of particular interest is the finding of electron-dense
deposits in the nucleolar vacuoles of the tumor cells. Similar
deposits have been found in Walker carcinoma cells (20) and in
cultured, human tumor cell lines (17, 27). The significance of
these deposits is, at present, not known. They may represent
unreduced osmium, uranyl, or lead salts (17). According to
Huxley and Zubay (11), uranyl acetate has a high affinity for
nucleic acids. It is possible that the electron-dense tumor cells
of this study, as well as cultured cell lines (17, 27), contain a
focal high concentration of nucleic acids in the electron-lucent
nucleolar vacuole, thus resulting in binding of metal stains.
ACKNOWLEDGMENTS
We thank Professor H. Swift for critical evaluation, particularly of
findings involving the nucleolar fine structure. The excellent assistance
of H. Patejak, D.D.S., and Mrs. M. James is sincerely appreciated. We
are also grateful to Mr. William R. Rennagel for editing this paper.
REFERENCES
1. Adams, W. R., and Prince, A. M. An Electron Microscope Study of
the Morphology and Distribution of the Intracytoplasmic
"Virus-like" Particles of Ehrlich Ascites Tumor Cells. J. Biophys.
Biochem. Cytol., 3: 161-170,1957.
2. Bekesi, J. G., Molnar, Z., and Winzler, R. J. Inhibitory Effect of
D-Glucosamine and Other Sugar Analogs on the Viability and
Transplantability of Ascites Tumor Cells. Cancer Res., 29:
353-359, 1969.
3. Bekesi, J. G., and Winzler, R. J. Inhibitory Effect of
D-Glucosamine and Other Sugars on the Biosynthesis of Protein,
Ribonucleic Acid and Deoxyribonucleic Acid in Normal and
Neoplastic Tissues. J. Biol. Chem.,244: 3766-3772, 1969.
4. Bekesi, J. G., and Winzler, R. J. The Effect of D-Glucosamine on
the Adenine and Uridine Nucleotides of Sarcoma 180 Ascites
Tumor Cells. J. Biol. Chem.,244: 5663-5668, 1969.
5. Birbeck, M. S. C., and Wheatley, D. N. An Electron Microscopic
Study of the Invasion of Ascites Tumor Cells into the Abdominal
Wall. Cancer Res.. 25: 490-497, 1965.
6. Fjelde, A., Sorkin, E., and Rhodes, J. M. The Effect of
Glucosamine on Human Epidermoid Carcinoma Cells in Tissue
Culture. Exptl. Cell Res., 10: 88-98, 1956.
7. Friedlaender, M., and Moore, D. H. Occurrence of Bodies within
1972
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1972 American Association for Cancer Research.
383
Z. Mainar and J. G. Bekesi
8.
9.
10.
11.
12.
13.
Endoplasmic Reticulum of Ehrlich Ascites Tumor Cells. Proc. Soc.
Exptl. Biol. Med., 92: 828-831, 1956.
Goldberg, B., and Green, H. The Cytotoxic Action of Immune
Gamma Globulin and Complement of Krebs Ascites Tumor Cells. I.
Ultrastructural Studies. J. Exptl. Med., 109: 505-510, 1959.
Herdson, P. B., and Kaltenbach, J. P. Fine Structural Changes in
HypotonicaJly Treated Ehrlich Ascites Tumor Cells. Exptl. Cell
Res., 42: 74-83, 1966.
Hruban, Z., and Rechcigl, M., Jr. Microbodies and Related Particles
(Morphology, Biochemistry, and Physiology). Intern. Rev. Cytol.
(Suppl. I), 1969.
Huxley, H. E., and Zubay, G. Electron Microscope Observations on
the Structure of Microsomal Particles from Escherichia coli. J. Mol.
Biol., 2: 10-18, 1960.
Johnson, J. M. A Study of Nucleolar Vacuoles in Cultured Tobacco
Cells Using Radioautography, Actinomycin D and Electron
Microscopy. J. Cell Biol., 43: 197-206, 1969.
Lynn, J. A., Martin, J. H., and Race, G. J. Recent Improvement of
Histological Technics for the Combined Light and Electron
Microscopic Examination of Surgical Specimens. Am. J. Clin.
Pathol.,45.- 704-713, 1966.
14. Molnar, Z. Ultrastructure of Calcifying Heart and Kidney. J. Cell
Biol., 27: 68A, 1965.
15. Quastel, J. H., and Cantero, A. Inhibition of Tumor Growth by
D-Glucosamine. Nature, 777: 252-254, 1953.
16. Recher, L., Briggs, L. G., and Parry, N. T. A Réévaluation
of
Nuclear and Nucleolar Changes Induced in Vitro by Actinomycin
D. Cancer Res., 31. 140-151,1971.
17. Recher, L., Whitescarver, J., and Briggs, L. The Fine Structure of
Nucleolar Constituents. J. Ultrastruct. Res., 29: 1-14, 1969.
18. Reynolds, E. S. The Use of Lead Citrate at High pH as an
Electron-opaque Stain in Electron Microscopy. J. Cell Biol., 17:
208-212, 1963.
19. Rubin, A., Springer, G. F., and Hogue, M. J. The Effect of
D-Glucosamine Hydrochloride and Related Compounds on Tissue
Cultures of the Solid Form of Mouse Sarcoma 37. Cancer Res., 14:
456-458, 1954.
20. Sankaranarayanan, K., and Busch, H. Dense Granules in Nucleoli of
384
21.
22.
23.
24.
25.
26.
27.
28.
Walker-256 Carcinosarcoma Cells of the Rat. Exptl. Cell Res., 38:
434-437, 1965.
Schoefl, G. I. The Effect of Actinomycin D on the Fine Structure
of the Nucleolus. J. Ultrastruct. Res., 10: 224-243, 1964.
Selby, C. C., Biesele, J. J., and Grey, C. E. Electron Microscope
Studies of Ascites Tumor Cells. Ann. N. Y. Acad. Sci., 63:
748-773, 1956.
Selby, C. C., Grey, C. E., Lichtenberg, S., Friend, C., Moore, A. E.,
and Biesele, J. J. Submicroscopic Cytoplasmic Particles
Occasionally Found in the Ehrlich Mouse Ascites Tumor. Cancer
Res., 14: 790-794, 1954.
St-Arneault, G., Walter, L., and Bekesi, J. G. Cytotoxic Effects of
Exogenous D-Galactosamine on Experimental Tumors. Intern. J.
Cancer, 7: 483-490, 1971.
Stenram, U. Autoradiographic, Biochemical, and Ultrastructural
Studies into the Effect of Actinomycin, 5-Fluorouracil, and
Adenosine on Nucleolar and Cellular Structure and Function. Nati.
Cancer Inst. Monograph, 23: 379-389, 1966.
Swift, H. Cytochemical Studies on Nuclear Fine Structure. Exptl.
Cell Res., Suppl. 9: 54-67, 1963.
Uzman, B. G., Foley, G. E., Farber, S., and Lazarus, H.
Morphologic Variations in Human Leukemic Lymphoblasts
(CCRF-CEM Cells) after Long-Term Culture and Exposure to
Chemotherapeutic Agents. Cancer, 19: 1725-1742, 1966.
Vasington, F. D., and J. V. Murphy: Ca~ Uptake by Rat Kidney
Mitochondria
and Its Dependence
on Respiration
and
Phosphorylation. J. Biol. Chem.,2J7: 2670-2677, 1962.
29. Watson, M. L. Staining of Tissue Sections for Electron Microscopy
with Heavy Metals. J. Biophys. Biochem. Cytol., 4: 475-478,
1958.
30. Wessel, W., and Bernhard, W. Vergleichende elektronenmikro
skopische Untersuchung von Ehrlich and Yoshida-Ascites Tumor
zellen. Z. Krebsforsch., 62: 140-162, 1957.
31. Yasuzumi, G., and Sugihara, R. A Comparative Electron
Microscopic Study on Ehrlich Ascites Tumor Cells, Yoshida
Sarcoma Cells, and Human Cancerous Peritonitis Ascites Cells.
Cancer Res., 18: 1167-1170, 1958.
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RESEARCH
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Effects of Amino Sugars on Ascites Tumor Cells
Figs. 1 to 4. Light micrographs showing the gross alterations caused by amino sugars in relation to control Ehrlich ascites tumor cells. The
unincubated control tumor cells had large, evenly shaped nuclei and distinct nucleoli and nuclear membranes (Fig. 1). In some cells, the cytoplasm
was finely vacuolated. The appearance of the cells of the control group incubated for 4 hr was essentially unchanged (Fig. 2). Incubation with
D-glucosamine for 1 hr resulted in coarse vacuolization of 95% of the cells (Fig. 3) and deformation of a large percentage of the nuclei. Nucleoli
were less conspicuous here than in the control groups. Incubation for 4 hr caused no significant further increase in cytoplasmic vacuolization, but
most nuclei were irregularly shaped and pycnotic, and the nucleoli were indistinct. Epon sections, stained with Mallory's azure 2-methyIene blue.
X 640.
Figs. 5 and 7. Electron micrographs of unincubated control Ehrlich (Fig. 5) and Sarcoma 180 (Fig. 7) ascites tumor cells. The nucleus (N)
contains a nucleolus with relatively tightly meshed, electron-dense components of the nucleolonema (small arrows) separating small compartments
of lower electron density. A nucleolar vacuole (v) is distinct. Membrane-limited bodies (lysosomes?) with a heterogeneous content (large arrows)
are numerous at the periphery of the Golgi zone (G). The matrix of the vesiculated mitochondria (M) contains an evenly distributed filamentous
material. Numerous virus-like particles are present within the cisternae of the RER (arrowheads).
Fig. 5. Electron micrograph of an electron-lucent Ehrlich ascites carcinoma cell of the unincubated control group, representing the majority of
the cells examined. X 17,500.
Fig. 6. Electron micrograph of a representative electron-dense Ehrlich ascites carcinoma cell of the unincubated control group. The nucleus (N)
is irregularly shaped and contains 2 nucleoli. Markedly electron-dense granules are present within the nucleolar vacuoles (v). One of the vacuoles
has a narrow opening towards the nuclear matrix (small arrows). The cytoplasm is tightly packed with organelles, mitochondria (M) flysosomes?)
(large arrows), and abundant RNP in the periphery of the cytoplasm. Virus particles are present in the RER (arrowheads). X 17,500.
Fig. 7. Electron micrograph of a representative Sarcoma 180 ascites tumor cell of the unincubated control group. X 17,500.
Fig. 8. Electron micrograph of a small perinuclear portion of an Ehrlich ascites carcinoma cell. As in the Sarcoma 180 tumor cells, the
cytoplasmic organelles are interwoven with a meshwork of fine filaments around the Golgi zone (G). The matrix of the mitochondria (M) and the
content of the cisternae of the RER are finely filamentous. Virus particles are also present in the RER (arrowhead). X 32,500.
Fig. 9. An area of the cytoplasm of an unincubated control Sarcoma 180 ascites tumor cell containing an aggregate of highly electron-dense,
ring-shaped particles with an electron-lucent center (arrowhead) lying free between a mitochondrion (M) and a segment of the RER
(ER). X 52,000.
Figs. 10 to 16. Electron micrographs of Ehrlich ascites carcinoma and Sarcoma 180 ascites tumor cells illustrating the time-dependent progress
of events secondary to incubation with amino sugars.
Fig. 10. Ehrlich ascites carcinoma cell treated for 15 min. The tightly compacted nucleolus contains a nucleolar vacuole (K), and at the
opposite surface an aggregate of the filamentous component of low electron density (arrowheads). Mitochondria (M) are swollen and lack the fine
filamentous matrix. Globular masses of RNP particles (small arrows) have formed in the cytoplasm, and vesicles of the Golgi zone are dilated (large
arrows). X 22,750.
Fig. 11. Ehrlich ascites carcinoma cell that has been incubated for 45 min with D-glucosamine, showing a compacted nucleolus with separation
of the filamentous component (arrowheads). The nucleolar vacuole (PO has an unusually wide surface contact with the nuclear matrix. The
peripheral cytoplasm in the lower portion of the picture is of much lower electron density than the perinuclear areas, which contain large
cytoplasmic vesicles (arrow). X 22,750.
Figs. 12 and 13. Small parts of Ehrlich ascites carcinoma cells that have been treated with D-glucosamine for 1 and 2 hr, respectively. They
illustrate the gradual decrease in the electron density of the filamentous component that accumulated at the periphery of the nucleolus
(arrowheads). These aggregates are free of clumps of interchromatin, which formed in the condensed nuclear matrix. The cytoplasm contains
numerous large vesicles (arrow). X 22,750.
Fig. 14. Ehrlich ascites carcinoma cell at the end of 3 hr of incubation with D-glucosamine, showing a granular, compacted nucleolus, which
typically lacks any electron-lucent filamentous components. The nuclear membrane is ill defined. Globular RNP aggregates (small arrows) are
present in the small rim of the cytoplasm, and a deep cytoplasmic invagination contains large vesicles (large arrow). X 22,750.
Fig. 15. Disintegrating Sarcoma 180 ascites tumor cell following 4 hr of incubation with D-glucosamine, representative of cells of both ascites
tumor lines. The nucleolus is tightly compacted and granular. The nuclear matrix is much diminished and appears disorganized, consisting of
clumped interchromatin. The narrow rim of cytoplasm of this cell and the adjacent one contains globular RNP aggregates (small arrows).
Mitochondria (M) contain small focal electron-dense deposits in the intercristal space (arrowheads). X 22,750.
Fig. 16. Ehrlich ascites carcinoma cell that has been treated with D-mannosamine for 4 hr, illustrating the advanced disintegration observed in
approximately 20% of the cells of both ascites lines after treatment with either of the amino sugars. The nucleolus has a coarse, granular
appearance, and the nuclear matrix (N) is coagulated into small clumps. Chromatin appears to exude into the cytoplasm through breaks in the
nuclear envelope (large arrows). The much-attenuated rim of cytoplasm contains mitochondria (M) with focal, electron-dense deposits. X 22,750.
Fig. 17. Representative Ehrlich ascites carcinoma cell following incubation for 4 hr with 2-deoxyglucose, showing preservation of the usual
structure of the nucleus (N) and nucleolus, including a nucleolar vacuole (v). The cytoplasm contains globular RNP aggregates and swollen
mitochondria (M). In about 20% of the tumor cells, mitochondria (M) had electron-dense focal deposits (arrowheads) consisting of small granules
in the intercristal space (insert). X 22,750. Insert, X 32,500.
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389
Effects of d-Glucosamine, d-Mannosamine, and
2-Deoxy-d-glucose on the Ultrastructure of Ascites Tumor Cells
in Vitro
Z. Molnar and J. G. Bekesi
Cancer Res 1972;32:380-389.
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