Download Virus Particles Associated with the

[CANCER
RESEARCH
29, 1313—1315. June 1969]
Brief Communication
Virus Particles Associated with the Transplantable
in the Rat'
Novikoff Hepatoma
Shuich
.@
i Karasaki
Laboratoires de Recherche, Institut du Cancer de Montréal,Hapital Notre-Dame et DIpartement d'Anatomie, UniversitI de Montréal,MontvIal,
Canada
Introduction.
hepatoma
virus
Electron
consistently
particle
in close
microscopy
of the Novikoff
reveals the presence
association
with
rat
of a characteristic
the plasma
membrane
of
tumor cells. Although the ultrastructure
of the fast-growing
anaplastic tumor has previously been described by several in
vestigators (5, 6, 8, 10), the occurrence of such particles had
not been reported in this tumor.
Materials and Methods.
Novikoff ascites hepatoma cells have
been maintained
in this institute by weekly intraperitoneal
transplantation
into male Sprague-Dawley
rats. Subcutaneous
inoculation
of the ascitic cells into the rats produces solid
tumors.
Both
the
ascitic
and
the
solid
forms
were
used
in the
present study. The specimens of tumor cells examined were
obtained from 15 implanted rats over the last three years. An
attempt was also made to isolate the particles from the ascitic
fluid according to the method of Dalton and Moloney (3) for
the purification of leukemic virus particles. All the tumor spec
imens and the particulate pellets were fixed successively with
buffered solutions of glutaraldehyde
and 0504 , dehydrated in
ethanol, and embedded in Epon. Thin sections were stained
with uranyl acetate and lead citrate and examined with a JEM
7A electron microscope.
Observations.
The fine structure of Novilcoff hepatoma cells
in both ascitic and solid forms was generally similar to that
reported previously by other groups (5, 6, 8, 10). In addition,
a characteristic
particle was found in the extra- or intercellular
spaces of all tumors examined. The particles tended to occur
singly and were localized adjacent or attached to the cell sur
face (Fig. 1). The number of such particles was generally one
to five per cell section, but up to 30 were found in some
sections. Similar particles were rarely noted in membrane
bounded vacuoles in the superficial cytoplasm (Fig. 2).
The particles were circular or slightly angular in profile with
an
average
diameter
of
approximately
100
mj.@. They
had
a
slightly eccentric nucleoid with a relatively electronlucent
in
termediate zone and were covered by an outer envelope which
had a triple-layered structure suggesting a unit membrane (Fig.
3). The outer surface was covered by a fringe of fibrillar mate
rial (Fig. 3). This envelope is essentially identical with the
1Supported
by grants from the National
Cancer Institute
Received January 15, 1969; accepted February 17, 1969.
of Canada.
plasma membrane in its organization. In some of the particles,
the nucleoid had a relatively electronlucent
central area (Fig.
4). A few of the particles exhibited a tail-like projection con
sisting,
in part,
of the envelope
material
(Fig.
5).
The cell surface also showed various forms suggesting that
particles are “budding out― from the plasma membrane. Such
a phenomenon
on the cell surface was frequently accompanied
by the formation
of numerous microvilli. Figs. 6 and 7 illus
trate
particles
presumably
in early
stages
of bud
formation.
Some of the incomplete particles are attached to the cells by
an elongated stalk end (Fig. 8). In the budding process, a
nucleoid component
appeared as a horse-shoe-shaped
layer of
high opacity which was located immediately under the plasma
membrane.
Sections of pellets obtained by high-speed centrifugation,
after preliminary
centrifugation
to remove ascitic cells, con
tained large amounts of the characteristic
particles (Fig. 9).
Their f'me structure was well preserved and identical to that
observed in the intact hepatomas although the particles inter
mingled with various cellular components.
Discussion.
By morphologic
criteria, the extracellular par
tides associated with Novikoff hepatomas are viral in nature.
The budding phenomenon
at the cell surface indicates that the
viruses are being replicated in and released by the tumor cells.
All the features of their ultrastructure,
distribution,
and devel
opment are identical to those of C-type virus particles as de
scribed by Bernhard (1). Except for certain leukemic condi
tions (1, 3, 4, 7, 9, 11), the presence of particles of this type in
rats is very rare (1). In the electron microscopic examination
of transplantable
rat hepatomas,
Dalton (2) reported a few
particles of C-type virus inside of cytoplasmic vacuoles only in
the fast-growing Morris hepatoma 3683. The present study has
demonstrated
that, in the fast-growing Novikoff hepatoma,
particles similar to those reported by Dalton occur more he
quently
vacuoles.
virus
in the extracellular
spaces
In the present
material,
particles
can
be readily
of
the
C-type
cell-free
obtained
by a technic for the purification
Viruses
as well as in the intracellular
group
of leukemic
have
fractions
in fairly
been
large
of intact
amounts
agents (3).
considered
as the
oncogenic agents in the murine leukemia, avian leukosis, and
Rous sarcoma ( 1). Therefore, the significance of virus particles
found in the fast-growing hepatomas may be of debatable im
portance in this neoplasia. Since the number of the particles
occurring in association with each cell has always been rela
JUNE 1969
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Research.
1313
Shuichi Karasaki
tively small, the viruses would appear to be replicating slowly
and continuously
in the cell without leading to cell death.
Persistent infection
of the hepatoma cells by the virus may
have been responsible for the rapid growth of these cells in the
host. In view of previous negative reports by several investi
gators (5, 6, 8, 10) examining earlier transplants of this tumor
line, the observation of viral particles in the one strain used in
the present investigation
suggests the late appearance
of a
masked viral genome during serial transplantation.
Alterna
tively, the agent could simply represent an unknown
non
pathogenic or murine leukemic virus which was picked up by
these cells as a passenger. The true nature of the particles,
however, must await further biologic characterization.
Acknowledgments.
The technical
assistance
of Mrs. Taeko
Karasaki
and Mrs. Michelle Robert is gratefully acknowledged.
A. J., and Moloney,
J. B. Recovery
of Virus from the
Blood of Rats with Induced Luekemia. In: R. J. C. Harris (ed.),
Interpretation of Ultrastructure, pp. 385—392. New York: Aca
demic Press Inc., 1962.
4. Dmochowski, L., Padgett, F., and Gross, L. An Electron Micro
scope Study of Rat Leukemia Induced with Mouse Leukemia Virus
(Gross). Cancer Res., 24: 869—899, 1964.
5. Howatson, A. F., and Ham, A. W. Electron Microscope Study of
Sections of Two Rat Liver Tumors. Cancer Res., 15: 62—69, 1955.
6. Hruban, Z., Swift, H., and Rechcigl, M., Jr. Fine Structure of
Transplantable
Hepatomas
of the Rat. J. Nail Cancer Inst., 35:
459—473, 1965.
7. Lapis, K., and Benedeczky,
I. Electron Microscopic Study of the
Shay Chioroleukemia.
Cancer Res., 27: 1544—1564, 1966.
8. Novilcoff, A. B. A Transplantable
Rat Liver Tumor Induced by
4-Dimethylaminoazobenzene.
Cancer Res., 1 7: 1010—1027, 1957.
9. Okano, H., Kunii, A., and Furth, J. An Electron Microscopic Study
of Leukemia
References
Induced
in Rats with Gross Virus. Cancer
Res., 23:
1169—1175, 1963.
1. Bernhard, W. The Detection and Study ofTumor Viruses with the
Electron Microscope. Cancer Res., 20: 712—727, 1960.
2. Dalton, A. J. An Electron Microscopical Study of a Series of Chem
ically Induced Hepatomas.In: P. Emmelot and 0. Mühlbock.(eds.),
Cellular
3. Dalton,
Control
Mechanisms
and Cancer,
pp. 211—225. Amster
dam: Elsevier Publishing Company, 1964.
10. Smetana, K., Unuma, T., and Busch, H. Ultrastructural
Nucleic Acids of Nucleolar
Granular Components
Studies on
in Novikoff
Hepatoma Cells. Exptl. Cell Res., 51 : 105—122,1968.
11. Weinstein, R. S., and Moloney, W. C. Virus-like Particles Associated
with Chloroleukemia
118: 459—461, 1965.
in the Rats.
Proc.
Soc. Exptl.
Biol. Med.,
Fig. 1. Part of an ascites hepatoma cell. Virus particles are seen either free or attached to the cell membrane. x 30,000.
Fig. 2. Virus particle in an intracytoplasmic vacuole (V) and intercellular space. x 30,000.
Figs. 3, 4. High magnification
(arrows).
of the particles
in Fig. 1. Both
the viral envelope
and the cell membrane
show a unit
membrane
structure
x 200,000.
Fig. 5. Extracellular particle with a tail-like process. x 200,000.
Figs. 6—8. Surface of the cell with incomplete
particles
budding
from the plasma membrane.
x 100,000.
Fig. 6. Early stage of viral budding.
Fig. 7. Viral bud protruding
from the plasma membrane.
Fig. 8. Viral bud at the tip of a long stalk.
Fig. 9. Section of a pellet prepared from the ascitic fluid. Isotonic citrate containing hyaluronidase was used in the isolation procedure.
Numerous particles are seen intermingled with various cellular components. x 20,000.
1314
CANCER
RESEARCH
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Research.
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1315
Virus Particles Associated with the Transplantable Novikoff
Hepatoma in the Rat
Shuichi Karasaki
Cancer Res 1969;29:1313-1315.
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