[CANCER RESEARCH 27 Part 1, 367-376,February 1967]
Nucleolar Changes in KB Tumor Cells Infected with Herpes Simplex
Virus
CARLO SIRTORI AND MARIA BOSISIO-SESTETTI
Department of Pathology of the National Cancer Institute,
and Laboratory of Electron Microscopy, Fondazione Carlo Erba, Milan, Italy
Summary
Cultures of KB tumor cells were infected with herpes simplex
virus [Bruxelles strain, infectious titer 2 X 106/ml tissue culture
infecting dose (TCIDÅ“)], and the resulting alterations were
studied by electron microscopy.
Peculiar lesions involve the nucleolus. In the cells containing
only a few immature virus particles in the nucleus, the nucleolar
ribosomes, the nonribosomic RNA filaments, and the amorphous
nucleolar component segregate and coalesce to form round
aggregates. Sometimes the fibrillar component seems to "cap"
the mass of the closely-grouped nucleolar ribosomes.
In the cells containing a greater number of virus particles,
both in the nucleus and in the cytoplasm, the nucleolar substances
aggregate and become more compact and electron dense. The
nucleolus decreases gradually in size until it fragments.
The earliest cytoplasmic change takes place as soon as the 1st
immature virus particles appear in the nucleus, and consists of a
decrease in ribosomes and polysomic chains. At more advanced
stages of viral invasion of the cell, numerous myelinic figures and
other nonspecific degenerative lesions appear in the cytoplasm,
preceding cellular lysis.
The ultrastructural changes of the nucleolus and the reduction
in polysomic chains are very similar to the alterations induced in
the cells by actinomycin D, mitomycin C, and 4-nitroquinolineA"-oxide.
Introduction
The cytopathic lesions caused by herpes simplex virus in
infected cells have been examined by several authors by light and
phase-contrast microscopy. These lesions consist mainly of
nucleolar changes, intranuclear inclusions, and syncytial forma
tions (1, 7, 13, 18). The various attempts made to demonstrate
by electron microscopy, the ultrastructural basis of the alterations
detectable in infected cells by the classic histology methods,
have so far produced uncertain data (23). In these last years,
many electron microscopy studies carried out on cells infected
with the herpes simplex virus have mainly elucidated problems
concerning morphology, development, and release of virus par
ticles (4, 8, 17, 31, 32). Only secondary importance, however,
has been given to the description and interpretation of the
changes caused by the virus in the ultrastructures of the host cell.
These changes, therefore, form the object of the present research,
in which the mode of action of the herpes simplex virus in infected
Received June 9, 1966; accepted September 23, 1966.
cells has been investigated by observation of the ultrastructural
changes preceding cell lysis.
Materials
and
Methods
Cells and Virus
Trypsinized KB tumor cells were grown in Leighton tubes in
Eagle's medium (3), containing 15 % rabbit serum and antibiotics
(streptomycin, 50 /ug/ml, and penicillin, 50 units/ml). When the
cells had multiplied to form a monolayer, they were infected with
herpes simplex virus (Bruxelles strain, infectious titer 2 x 106/ml
TCTD5C).The infecting dose was 0.2 ml/tissue culture, containing
200,000-300,000 cells.
Electron Microscopy
Specimens of uninfected KB cells and KB cells 18, 24, 42, 48,
and 72 hr after infection were incubated for 10 min at 37°C
with a 0.25% buffered trypsin solution (1 ml/10 ml of culture
medium), to detach the cells from the glass.
The cells were then suspended in the culture medium and
treated as follows: short prefixation by addition of a few drops of
1% osmium tetroxide (15); low-speed centrifugation for 2-3 min;
further fixation of the sediment with 1% osmium tetroxide for
30 min; embedding in Vestopal W; staining of thin sections, cut
with an ultramicrotome Ultrotome LKB, with uranyl acetate
(1% aqueous solution) and lead citrate (21); observation with a
Hitachi-HS-7 electron microscope.
Results
Uninfected KB Cells
The majority of the uninfected KB cells have large nuclei,
which frequently are round or oval, and less often pleomorphic
or lobate. They contain little chromatin and 1 nucleolus, very
often located at the periphery. Under the electron microscope,
the nucleolus appears to be formed of dense ribonucleoprotein
(RNP) granules (also termed "nucleolar ribosomes") of 100-150
A diameter, irregularly scattered on a loosely meshed network
constituted by an electron-dense, finely fibrillar substance
(nucleolonema). In some of the meshes of the nucleolonema an
amorphous and less electron-dense nucleolar component can be
observed (Fig. 2).
The cytoplasm of the uninfected KB cells (Fig. 3) contains
some irregularly placed ergastroplasmic cisterns, many mito
chondria of different shapes and sizes, and numerous very elec
tron-dense osmiophilic bodies, which probably consist of phago-
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367
Carlo Sirtori and Maria Bosisio-Bestetti
cytized material. The cytoplasm appears very rich in ribosomes,
scattered and arranged in clusters and polysomic chains (Fig.
3a). The Golgi apparatus and the smooth endoplasmic reticulum
are poorly developed. The KB cells cultured in monolayer tend
to assume an epithelial-like disposition (Fig. 1). On the cell
boundaries occur numerous desmosomes, and several rows of
pinocytotic vesicles can be observed near the free surface of the
cells.
KB Cells after Infection with Herpes Simplex Virus
The KB cells were examined 18, 24, 42, 48, and 72 hr after
infection. In the specimens fixed 18 and 24 hr after infection, the
cells containing virus particles are very few and most of the cells
appear quite undamaged. The few cells which appear invaded by
virus contain a variable number of virus particles, ranging from
a very few immature ones in the nucleus to a great number both
in the nucleus and in the cytoplasm.
In the specimens fixed 42 hr after infection, all the cells contain
virus. Also in this case, however, the number of intracellular viral
particles is extremely variable. Therefore, the only difference
between the specimens examined 18 and 24 hr after infection and
42 hr after seems to be in the number of cells containing the
virus, while the different degrees of viral invasion of the cells and
the consequent ultrastructural changes look identical. For this
reason, in the description of these alterations, reference is made
mainly to specimens examined 42 hr after infection, where it is
much easier to find many different degrees of viral invasion in
the same section.
Forty-eight and 72 hr after infection, the great majority of
cells are overloaden with virus particles and show severe and non
specific alterations of cellular lysis. In these specimens, cells
containing only a very few or no virus particles at all are no
longer observed.
The most important and frequent ultrastructural change
observed in KB cells containing herpes simplex virus particles
affects the nucleolus.
As soon as a few immature virus particles appear in the nucleus,
the nucleolar substances seem to change their distribution. They
segregate from one another and coalesce to form compact, round
masses, and the nucleolar appearance varies according to the
arrangement of the nucleolar components aggregates. In the
nucleolus, a central aggregate of closely grouped RNP granules
and 1 or 2 lateral electron-dense masses showing a finely fibrillar
structure can be clearly observed (Fig. 4). In contact with these
zones, there are also small aggregates of amorphous and more
electron-dense material (Fig. 4). Sometimes, a vacuole containing
scanty scattered material appears in the nucleolus, and the
fibrillar substance seems to cap the zone of RNP granules (Fig. 5).
Further nucleolar changes follow virus multiplication in the cell.
The mass of RNP granules becomes more compact and in contact
with it 1 or more round buds appear. They are very electron
dense and compact; therefore it is difficult to distinguish whether
they consist of amorphous or fibrillar nucleolar substance (Figs.
6, 7). These buds sometimes seem to disaggregate into minute
fragments (Fig. 7) and at other times to detach themselves from
the granules (Fig. 8) to remain isolated in the nucleus. When
the virus particles become more numerous in the nucleus and
368
begin to appear also in the cytoplasm, the nucleolar size decreases
(Fig. 9) and the nucleolar substance aggregates become more
compact. This makes it still more difficult to distinguish their
composition clearly.
In the cells where the nucleus is occupied by a large amount of
virus particles in crystalline array, the nucleolus is reduced to
small scattered debris or is no longer visible.
The evolution of the nucleolar damage is accompanied by a
progressive reduction in the chromatin. This latter collects at the
periphery of the nucleus and adheres to the inner membrane.
The earliest change in the cytoplasm, occurring nearly simul
taneously with the appearance of the 1st virus particles in the
nucleus, is an apparent reduction in all the cytoplasmic organules
(rough endoplasmic reticulum, mitochondria, osmiophilic bodies),
especially in the ribosomes which tend to be scattered rather than
grouped in clusters or polysomic chains. When the virus begins
to pass from the nucleus into the cytoplasm, the latter undergoes
degenerative changes. Numerous myelinic figures (Fig. 10)
appear, some of which seem to derive from the rough endoplasmic
reticulum. Forty-eight and 72 hr after infection, all the cellular
structures appear severely damaged by the massive viral invasion.
The nucleus is almost lacking in chromatin and contains a great
number of immature virus particles in crystalline array (Fig. 11).
The nucleolus is no longer visible. All the cytoplasmic structures
show severe and nonspecific alterations, usually observed in
cellular lysis from any cause.
Discussion
The most recent histochemical and ultrastructural researches
on the composition of the nucleolus (14) show that it consists of
3 fundamental components: RNP granules or nucleolar ribo
somes, fine fibrils probably constituted by nonribosomic RNA,
and an amorphous electron-transparent material. The process of
sorting out and coalescence of these nucleolar substances to form
nucleolar "caps," "buds," and "satellites," accompanied by a
decrease in scattered ribosomes and especially in polysomic
chains in the cytoplasm, seems to be the earliest and probably
most characteristic lesion caused by the herpes simplex virus in
KB cells.
Although the growth cycle of the herpes simplex virus is of
the order of 12 hr, only a few cells were seen to contain virus
particles in the samples examined 18 and 24 hr after infection.
This may lead one to think that the observations made over a
period of 18-72 hr apply to a continuing infection with an initial
low inoculum. In this case, the possibility could arise that the
observed alterations of KB cells were due to some side effects,
rather than directly to the infection. Under our experimental
conditions, however, the number of virus particles per host cell
was at least 1, and a marked cytopathic effect was observed under
the phase-contrast microscope in cell specimens examined 12 and
18 hr after the infection. For this reason, we think that the small
number of virus-containing cells seen in the samples observed
under the electron microscope 18 and 24 hr after the infection
may be explained by the 2 following factors: (a) the thin sections
examined may have been cut through a portion of the cell where
there were no virus particles; (6) since the examined cells were
CANCER RESEARCH VOL. 27
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Changes in Tumor Cells Infected with Herpes Simplex
not a homogeneous done, some of them may have been less
sensitive to the infection.
Nucleoli with morphologic alterations similar to those observed
have been described in cells treated with actinomycin D both
in vitro (25) and in vivo (2, 10, 29), with mitomycin C (12), and
with 4-nitroquinoline-Ar-oxide (16, 22). The findings most similar
to ours are those obtained in vivo by Jézéquel
and Bernhard (10)
in the exocrine pancreas cells of mice treated with actinomycin D
and examined at various intervals after administration of the
drug. The ultrastructural changes induced in the pancreas cells
by actinomycin D could, at least where the nucleolus is concerned,
represent the morphologic appearance of its action on the DNAdependent synthesis of ribosomic and messenger RXA, through
mechanisms recently demonstrated by biochemical researches
(5, 11, 19).
The observation that the redistribution of the nucleolar com
ponents takes place only in cells treated with agents which modify
nucleic acid metabolism [e.g., actinomycin D (5, 11, 19), mito
mycin C (9, 20, 26), 4-nitroquinoline-.V-oxide (22), and others
(27)] justifies the regarding of this morphologic picture as specific
for alteration of nucleic acid metabolism at different levels (27).
Our own research findings show that the lesions produced by the
herpes simplex virus in KB cells involve at first only nucleolar
and cytoplasmic structures containing RNA. These results and
the above data obtained from literature could lead one to assume
that also the redistribution of the nucleolar components sub
sequent to herpes simplex virus infection might represent the
morphologic manifestation of an action of the virus at some un
known level of nucleic acid metabolism in the host cell. Recent
researches on the metabolism of nucleic acids in animal cells
infected with herpes simplex virus (11, 24, 30), show, however,
that the synthesis of at least a fraction of DNA-dependent RNA,
i.e., the transfer RNA of the host cell, is not inhibited by the
virus (30). It is evident, therefore, that the mechanism by which
the morphologic changes are induced in the nucleolus of infected
cells is still an unknown biochemical phenomenon.
Observation of the subsequent stages of nucleolar damage
(size reduction, segregation, and fragmentation of nucleolar
components) show that this occurs at equal rate with an apparent
decrease of ribosomes and in particular with the reduction of the
polysomic chains. Nevertheless, while the nucleolus undergoes
rapid changes to the point of being reduced to small fragments,
and then virtually disappearing, a certain amount of scattered
ribosomes and a few polysemes always remain in the cytoplasm.
This finding is difficult to interpret. It could be due to a more
rapid turnover of nucleolar RNA compared with that of the
cytoplasmic RNA, or to a passage of nucleolar RNA into the
cytoplasm (5). Study of the subsequent stages of nucleolar
involution shows frequently that the zone of RNP granules
decreases in size earlier than the masses of the other nucleolar
components. This finding seems in accordance with both the
hypotheses put forward.
The appearance of myelinic figures in the cytoplasm, following
massive viral invasion of the cell, should be considered an entirely
nonspecific ultrastructural lesion, since myelinic figures may be
observed commonly in cells undergoing lysis from any cause.
The observation that the herpes simplex virus induces great
morphologic alterations in KB tumor cells raises the question on
the opportunity of further investigations of the oncolytic action
of this virus. This, despite the fact that some attempts to employ
herpes simplex virus (6, 28) as an oncolytic agent have up till
now given doubtful or negative results.
Acknowledgments
The authors gratefully acknowledge the assistance of Dr.
Romano Angelucci from the Department of Virology of the Carlo
Erba S.p.A., Milano, in the tissue and virus culture work.
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FIG. 1. Monolayer of uninfected KB cells, showing an epithelial-like disposition. The cytoplasm is very rich in ribosomes and con
tains numerous mitochondria and osmiophilic bodies. On the cell boundaries there are some desmosomes.
X 15,000.
FIG. 2. Nucleolus of an uninfected KB cell: a, zone of ribonucleoprotein
granules; 6, fine fibrillar substance (nucleolonema); c,
amorphous substance; N, nucleus.
X 34,000.
FIG. 3. Cytoplasm of an uninfected KB cell, containing a great number of ribosomes both scattered and grouped to form polysomic
chains. Note numerous pinocytotic vesicles at the free surface of the cell. X 34,000. a, detail of Fig. 3. Clusters of ribosomes and poly
somic chains. X 68,000.
FIG. 4. Nucleolus of a KB cell 42 hr after the infection with herpes simplex virus. The cell contains only a few immature virus par
ticles in the nucleus. The nucleolar components have coalesced into several zones, a, ribonucleoprotein granules zone; 6, zone of co
alesced nucleolonema; c, amorphous substance.
X 41,000.
FIG. 5. Same condition as shown in Fig. 4. The nucleolonema (6) seems to "cap" the mass of ribonucleoprotein
granules (a). Note
a vacuole between the 2 aggregates of nucleolar substance and a "satellite" (s) which seems to derive from zone 6. c, amorphous sub
stance; v, virus particle.
X 41,000.
FIG. 6. More advanced nucleolar lesion in a KB cell. The cell contains several virus particles both in the nucleus and in the cytoplasm.
The central zone of the nucleolus, formed by closely-packed ribonucleoprotein granules (a), is surrounded by very electron-dense, round
masses, probably derived from coalesced nucleolonema (6). v, virus particles.
X 63,000.
FIG. 7. Same condition as shown in Fig. 6. One of the electron-dense masses of coalesced nucleolonema (6) appears to disaggregate
into small fragments and spread throughout the nucleus, a, ribonucleoprotein
granules.
X 63,000.
FIG. 8. Same condition as shown in Figs. 6 and 7. The ribonucleoprotein
granules (a) have detached from the coalesced nucleolo
nema (6). v, virus particles.
X 41,000.
FIG. 9. KB cell containing a great number of virus particles scattered both in the nucleus and in the cytoplasm. The nucleolar size
appears much reduced. Note the paucity in chromatin of the nucleus, v, virus particles.
X 41,000.
FIG. 10. Cytoplasm of a KB cell 48 hr after the infection. In comparison with Figs. 1 and 3, the number of ribosomes is markedly
reduced. Several myelinic figures and virus particles appear in the cytoplasm. The mitochondria are alterated and more electron dense.
f, virus particles; A', nucleus.
X 54,000.
FIG. 11. Nucleus of a KB cell 72 hr after the infection. The nucleus is almost entirely occupied by a conspicuous amount of immature
virus particles in crystalline array. Note some chromatin still present at the periphery. N, nucleus.
X 63,000.
370
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VOL. 27
Nucleolar Changes in KB Tumor Cells Infected with Herpes
Simplex Virus
Carlo Sirtori and Maria Bosisio-Bestetti
Cancer Res 1967;27:367-376.
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