1961;21:193-197.

Cancer Res

S. Dales and A. F. Howatson

Virus-like Particles in Association with L Strain Cells

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Virus-like Particles in Association with L Strain Cells
S. DALES*ANDA. F. HOWATSON
(Division of Biological Research, Ontario Cancer Institute, Toronto, Canada)
SUMMARY
Electron microscope studies of three sublines of Earle's L strain cells showed that
in two of the three sublines virus-like particles of type C were present within cyto-
plasmic vacuoles and outside the cells close to the cell surface. The particles formed by
a budding process at the ceil membrane. Similar particles were observed in pellets
prepared from the medium in which the cells had been grown, and in cells of tumors
produced in C3H mice by inoculation of the cultured cells. A second type of particle,
type A, was observed within cytoplasmic vacuoles in all three sublines of cells culti
vated in vitro. No biological activity has so far been associated with either type of
particle.
Since methods for preparing cells and tissues for
thin sectioning and electron microscopy have be
come available, considerable effort has been ex
pended in searching for structures that might be
identified as viruses within and around cells of
many different types of tumors. In tumors of
known viral etiology these efforts have been for
the most part successful in that particles pre
sumed to be the causative agents have been ob
served in many instances (3), although it should
be noted that in only a very small number of these
has the further step required for unequivocal
identification been made, namely, the establish
ment of a correlation between the number of par
ticles of the type observed and tumor-inducing
activity (25). In a number of tumors for which
there is no biological evidence of viral origin, struc
tures morphologically similar or identical to those
observed in certain virus-induced tumors have
been described (3). The significance of such "virus-
like" particles is difficult to assess in the absence of
any demonstrable biological activity. Neverthe
less, it cannot be assumed that their presence in
and around tumor cells is fortuitous, and it is of
some importance to determine their relation to the
tumor cells and frequency of occurrence in differ
ent tumors. This is especially true in view of cur
rent investigations of the possible viral etiology of
human tumors in which morphological observa
tions may be important, since many experimental
* Present address: Department of Cytology, The Rocke
feller Institute, New York 21, N.Y.
Received for publication July 22, 1960.
approaches used with animals are not feasible in
human beings.
In the present report two types of virus-like
(VL) particles that have been observed in associa
tion with certain lines of Earle's L strain cells are
described, and their significance is discussed. The
origin and relative malignancy of the cell lines
used are described in the following section.
MATERIALS AND METHODS
Derivation of cell lines.Earle's strain L cells
were originally derived in 1943 from an expiant
of subcutaneous connective tissue which had been
treated in vitro for several months with methyl-
cholanthrene (8). Upon injection into adult C3H
mice these cells were found to be highly malignant,
producing sarcomas in 68 per cent of the mice in
jected. After repeated passage of the cells in vitro
this high incidence of tumors declined to only 1 per
cent. In 1948 L cells were cloned by Sanford et al.
(20), and a permanent line of cells, the NCTN 929
line, was established; this was found to produce
tumors in a small percentage of adult C3H mice.
However, the tumor incidence in adults could be
increased from 1 to 65 per cent by irradiating the
hosts before injection (9), and more recently it was
shown that the incidence of tumors produced by
injecting newborn mice was very high (19). These
results showed that the NCTN line of L cells was
still highly malignant, although the adult animals
had apparently acquired some immunity against
these cells. Several sublines of the clone NCTN
929 have been developed, three of which were used
193
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194 Cancer Research Vol. 21, February 1961
in the present investigation. These sublines have
been designated L2, AMK2_2, and L4CZ. The deri
vation and characterization of the first two of these
sublines of L cells have been described in detail
(19). Briefly, the 1,2 cells were obtained from a
stock of frozen L cells which had been kept at
70 C. for several months; the AMK2_2 cells
were derived from a clone obtained by dilution and
plating of the AMK subline of cells (which is
karyologically almost identical with the L2 sub-
line); L4CZ cells were recently derived from L2
cells by Axelrad and Evans1 in the following way.
Suspensions of L2 cells were injected into newborn
C3H/HefOci mice. Cells of a tumor which grew
from these injected cells were then re-injected into
2-3-month-old mice of the same strain. A second
transplant tumor arising in one of these animals
was again transplanted. After dispersal by tryp-
sinization, the cells of this tumor grew in suspen
sion in the same way as the L2 and AMK sublines.
Although karyotypically L4CZ cells are very simi
lar to the L2 cells, they are much more malignant,
having retained their high malignancy after many
generations of culturing in vitro and several pas
sages in vivo. About 44 per cent of C3H mice given
injections at birth of IO2 L4CZ cells develop tu
mors, whereas only 4 per cent of the mice which
receive a similar dose of L2 cells develop malignant
growths.
Specimen material.Electron microscope
studies were made on three types of speci
men: (a) cells of each of the three sublines culti
vated m vitro, (b) tumors that resulted from giv
ing injections to newborn C3H mice of cells of the
three sublines, and (c) pellets prepared from the
nutrient media in which the cells had been grown.
The cells were propagated in vitro in suspension
culture by the technic of Siminovitch et al. (22),
the nutrient medium being CMRL 1066 (11)
supplemented with 10 per cent horse serum. These
cells and also the tumors that arose from them in
mice were prepared for thin sectioning and elec
tron microscopy by standard procedures. The pel
lets were prepared in the following way. About
1-2 X IO7 cells, sedimented by low-speed cen-
trifugation, were suspended in 30-50 ml. of fresh
nutrient medium and were incubated in suspen
sion for approximately 24 hours. On the following
day the cells were again sedimented and were once
more suspended in fresh medium and incubated.
The supernatant fluids obtained after the removal
of cells following each 24-hour period of culturing
were processed separately. Each sample of fluid
was centrifuged at an intermediate speed
(20,000 X g for 15 minutes) to remove some of
1Unpublished experiments.
the cell debris. The supernatants were then cen
trifuged 104,000 X g tor 60 minutes. The pellets
formed were resuspended in 20 ml. of saline solu
tion with 0.125 per cent trypsin added and were
incubated for 30-60 minutes at 37 C. to digest
some of the remaining protein material. Pellets
prepared by a final centrifugation at 104,000 X g
for 60 minutes were processed for electron micros
copy in the same way as the cells and tumor tis
sue. To enhance the contrast of thinly sectioned
specimens two procedures were employed. In the
first, specimens were "stained" for 20 minutes
with a 1 per cent solution of uranyl acetate dis
solved in 70 per cent alcohol prior to further de
hydration and embedding. In the second, thin
sections were "stained" with lead hydroxide by
the procedure of Watson (24).
RESULTS
The components of fine structure generally ob
served in cells of the fibroblast type were present
in Earle's L cells. Thus, in the cytoplasm of thinly
sectioned cells there were numerous oval and
elongated mitochondria, many smooth- and rough-
walled vesicles of varying size, ribonucleoprotein
granules dispersed throughout the cytoplasm, and
fine processes emanating from the cell membrane.
Examination in the electron microscope of thinly
sectioned L cells of the L2 and AMK sublines,
which had been cultured in vitro, revealed in addi
tion to normal components the presence of virus-
like (VL) particles attached or close to the cell
membrane. These occurred in 10-20 per cent of the
cells examined, usually singly or in small groups;
but occasionally larger aggregates were observed,
such as the one illustrated in Figure 1. Most of
these particles were spherical and ranged in size
from 80 to 110 m/j, but a few were as large as 180
m p in diameter. Generally, each particle contained
a centrally placed nucleoid surrounded by a less
dense zone and a limiting membrane, but occa
sionally particles devoid of dense centers were also
observed. The nucleoid was variable in density and
appearance, sometimes being uniformly dense but
more often consisting of a dense rim with a less
dense, usually filamentous interior. This varia
bility might be due to the presence of incomplete
forms of these particles or could result from differ
ences in the quality of preservation and staining.
Similar particles were less frequently observed
either singly or in groups within membrane-bound
cytoplasmic vesicles. Figure 2 shows an area of
cytoplasm in which there are a number of vesicles,
two of which contain groups of VL particles. Most
of these particles fit the morphological category
"type C," proposed by Bernhard (6). Protuber-
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DALESANDHOWATSONVirus-like Particles icith L Strain Cells
195
anees of the cell membrane or the membrane sur
rounding cytoplasmic vacuoles were commonly ob
served at, or close to, the sites where VL particles
were present. The protuberances were limited on
the outside by a membrane that in many instances
appeared to be continuous with the cell membrane.
On the inner surface there was an accumulation of
dense amorphous material and a second very dense
membrane. In Figures 3 and 4 are shown what are
interpreted as stages in the formation of VL par
ticles; the protuberance in Figure 3 (right) repre
sents an early stage; in Figure 4 the "budding"
process is more advanced, and in Figure 3 (left)
is shown a particle apparently almost separated
from the cell. This particle differs from typical
type C particles in that the center is less dense and
the surrounding zone denser. Particles similar to
the one illustrated were found among type C par
ticles close to the cell membrane and also within
cytoplasmic vacuoles. They may represent free
particles which are incompletely developed. It is
likely, however, that some of these represent
protuberances that have been sectioned parallel
to the cell surface.
Occasionally finger-like projections much longer
than the protuberances already described were ob
served at the cell surface at locations where VL
particles were present or forming. These were of a
diameter approximately the same as that of the
VL particles. One ot these projections is shown in
Figure 5. Apart from its greater length (approxi
mately 400 mju) it is similar in structure to the
much commoner short protuberances, suggesting
that it is closely related to them.
In addition to the particles and projections from
the membrane that were present in cells of the L2
and AMK sublines, there were in all three sublines
cultured in vitro smaller particles of a different
morphology. These were usually distributed singly
within cytoplasmic vesicles. They measured 60-80
mu in diameter and had a center of low density
surrounded by a double-layered membrane. Fig
ure 7 shows a portion of the cytoplasm of an L2
cell in which there are cytopiasmic vacuoles con
taining particles of the second type, indicated by
arrows. These particles are of type A, according
to Bernhard's classification (3). In the same fig
ure, near the surface of the cell, are shown a few
particles of the first type described, type C.
Many of the type C particles appeared to be
separated from the cells. To test whether they
were released into the nutrient medium, super
natant fluid from ceil cultures was centrifuged
into pellets, as previously described. Examina
tion of thinly sectioned pellets in the electron
microscope routinely showed the presence of
large numbers of particles identical to the type C
particles seen in sectioned cells. A typical area of
a sectioned pellet is shown in Figure 8. A small
group of VL particles is shown in Figure 6 at higher
magnification. These type C particles were seen
only in pellets prepared from supernatant fluids
obtained from cultures of L2 and AMK cells. No
type A particles were seen in these sectioned pel
lets. Neither type A nor type C particles were
found in pellets of supernatant fluids prepared
from L4CZ cultures, nor in pellets prepared from
fresh nutrient medium.
TABLE1
SUMMARYOFOBSERVATIONS ONTHE
OCCURRENCE OFVLPARTICLES
MATERIAL EXAMINEDSolid
tumors produced by
sublines AMK and LjSolid
tumors produced by
line L4CZCells
of sublines AMK and
LI cultured in suspensionCells
of subline L4CZ cul
tured in suspensionPellets
from nutrient medium
in which L2and AMK cells
had been grown for 24
hoursPellets
from nutrient medium
in which L4CZ cells had
been grown for 24 hoursPellets
from fresh nutrient
mediumLOCATION
ANDTTPE or PARTICLESType
ACytoplasmic
vacuolesCytoplasmic
vacuolesCytoplasmic
vacuolesCytoplasmic
vacuolesNot
foundNot
foundNot
foundTyp
CCytoplasmic
vacuoles cell
membraneNot
foundCytoplasmic
vacuoles cell
membraneNot
foundPresentNot
foundNot
found
Examination of the tumors grown in C3H mice
showed that particles of type C, identical in ap
pearance to those found in cells cultured in vitro,
were present in tumors induced by L2 and AMK
cells but were absent in tumors induced by the
liiCZ subline. However, tumors induced by all
three sublines of cells contained type A particles.
The observations on sectioned cells and pellets
have been summarized in Table 1.
DISCUSSION
We shall consider first the type C particles.
These particles have the general morphological
characteristics that are associated with many
known viruses including several tumor viruses,
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196 Cancer Research Vol. 21, February 1961
namely, a dense central body or nucleoid surround
ed by a less dense zone, the whole being enveloped
in a dense limiting membrane. It is possible that
they represent the etiological agent that was re
sponsible for the original conversion of the cells to
malignancy, but there is no evidence in support of
this; and it would seem unlikely, since one subline
did not contain any particles. Nevertheless the
particles may represent tumor virus. There are
now several instances of the recovery from tumor
tissue of tumor virus of a type apparently unre
lated to the agent causing the tumor from which
it was recovered. FOPexample, polyoma virus has
been recovered from leukemic tissue (23) and from
mammary tumor (16) of the mouse in the induc
tion of which other viruses, both distinct from
polyoma, are implicated. Again, a virus causing
leukemia has been obtained from a sarcoma for
which there is no evidence of viral origin (17).
Thus it would seem that tumor viruses may be
carried as passenger viruses and that tumor cells
provide a suitable environment for them. Further,
the mode of formation of the particles that we ob
served around L cells, namely, a budding process
at the cell membrane, closely resembles that de
scribed for certain tumor viruses, the identity of
which is reasonably well established (3). The most
detailed studies have been made by deHarven (10)
on the leukemia virus of Friend. Thus, from a
purely morphological standpoint, the VL particles
are indistinguishable from known tumor viruses
both in structure and in mode of formation. How
ever, as already pointed out, the significance of
such morphological evidence is open to question
in the absence of demonstrable biological activity.
To test whether the particles released from the
two strains of L cells had any biological, particu
larly tumor-inducing, activity, pellets of the par
ticles obtained as described were resuspended in
buffered saline solution and injected subcutane-
ously into newborn CSHmice and Syrian hamsters.
To date, 8 months later, no biological activity of
these preparations has been detected. These neg
ative results do not, of course, rule out the pos
sibility that the VL particles are in fact tu
mor viruses, for it is known that the action of some
tumor viruses is strongly conditioned by host fac
tors, and the conditions required to demonstrate
biological activity may not have been satisfied in
the limited tests that we have carried out.
Another possibility is that the VL particles are
a nontumor passenger virus. Of nontumor viruses,
the myxoviruses probably bear the closest re
semblance to the VL particles in size, appearance,
and mode of formation (2, 18). However, injec
tion of suspensions of VL particles into mice pro
duced none of the symptoms associated with
myxoviruses, and inoculation onto cultures of
mouse embryo cells had no apparent effect. Fur
ther, the presence of the VL particles in no way
affected the susceptibility of L cells to infection
by vesicular stomatitis virus, whereas L cells in
fected with myxoviruses are not susceptible when
subsequently exposed to vesicular stomatitis virus
(12). Thus, there is no evidence that the VL par
ticles are related to the myxovirus group. At
tempts to induce the production of VL particles
in the JtCZline by inoculating cultures of these
cells with suspensions of particles obtained from
the other two lines were not successful. Thus, all
our attempts to associate biological activity with
the type C particles have failed, and there is no
evidence other than their strong resemblance in
structure and mode of formation to known viruses
that the particles are viral in nature.
A remaining possibility is that the particles are
not infectious units but are a product of some kind
of secretory process in the cells. This presumed
secretory activity did not seem to disturb cellular
function, for the lines of cells that produced par
ticles grew as rapidly and appeared as healthy as
the one that did not. Further, particle production
was not influenced by environmental factors, since
the two lines that produced particles did so wheth
er grown in vitro or in vivo.
The viral nature of the type A particles is also
questionable. It appears that they are not released
from the cells as are the type C particles, because
they were not present in the pellets prepared from
the nutrient medium in which the cells were
grown. It was therefore not possible to test the
type A particles for biological activity in the
same way as the type C particles. Their claim to
be considered as possible viral agents rests on
purely morphological grounds. Particles of type A
have been described in cells of many different
types of tumor in mice. They may occur sparsely
or in large aggregates in tumors of known viral
etiology such as mouse mammary tumors (4, 5),
or they may be present in tumors for which viral
etiology has not been demonstrated (15). It has
been suggested that they represent a stage in the
formation of mature virus particles (3), but they
may equally well be a reaction of the cell to the
presence of virus or to some other abnormal condi
tion. Although in some tumors a close association
has been observed between the Golgi region,
known to be concerned in secretory activities of
the cell, and type A particles (5, 15), it is not evi
dent in L cells. Nevertheless, the possibility that
type A particles in L cells represent some abnormal
type of secretion should not be overlooked. Type A
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Fio. 1.Part of an Lj cell from a suspension culture. A large
number of type C particles are shown in association with the
cell membrane. X 44,000.
FIG. 2.Portion of the nucleus (upper right) and cytoplasm
of an LScell from a suspension culture, showing two groups
of type C particles within cytoplasmic vacuoles. X 44,000.
FIGS. 8-5.These Figs, illustrate stages in the formation
of type C particles at the cell membrane.
FIG. 8.Within both the protuberance of the membrane
and a circular structure which lies in close proximity to it,
there is a dense inner membrane. The circular structure may
be an incompletely developed type C particle, or it may be
the cross section through another protuberance which was
oriented perpendicularly to the plane of section. X 128,00(1.
FIG. 4.Amicrovillus with a VL particle forming at its
tip. Note the dense material closely applied to the tip of the
microvillus and the very dense semicircular inner membrane.
XI 28,000.
FIG. 5.Afinger-like projection at the surface of an L
cell. Apart from its greater length, its morphology is similar
to that of the structures shown in Figs. 3 and 4. X 128,000.
FIG. 6.Detail of type C particles, from a sectioned
pellet similar to that shown at lower magnification in Fig.
8. X200,000.
FIG. 7.Area in the cytoplasm of an Lj cell illustrating
type A particles (indicated by arrows). Each has a double
limiting membrane and a center of low density. At the upper
right of the micrograph is a group of type C particles near the
cell membrane. X38.000.
FIG. 8.Area of a sectioned pellet of partially purified
VL particles. X38.000.
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DALES ANDHOWATSONVirus-like Particles with L Strain Cells
197
particles seem to be associated with neoplasia, for
which they have not to our knowledge been de
scribed in non-neoplastic cells; but their role and
significance, if any, in .the neoplastic process re
mains to be determined.
There is now a long list of tumors both in ani
mals and in human beings in association with
which particles suspected of being virus have been
observed, but for which no viral activity has been
demonstrated biologically. These include Ehrlich
ascites tumor cells (1, 21), plasma-cell tumors (15),
human leukemic lymph nodes (7), and mammary
tumor (14), and uterine epithelioma (13) in the rat.
Whereas such observations are of interest and
may be of great significance, it is clear that more
evidence must be acquired before any claim to a
role in carcinogenesis can be attributed to particles
because of their resemblance to known viruses.
The present studies show that, even when it is
possible to isolate the "virus-like" particles in
fair quantity and in fairly pure state, it may still
be difficult to obtain evidence that they have any
biological activity.
ACKNOWLEDGMENTS
Tins investigation was carried out during the tenure by one
of the authors (S. Dales) of a post-doctoral fellowship of the
National Cancer Institute of Canada. It was supported by
grants from the National Cancer Institute of Canada and the
Foster Bequest Fund of the University of Toronto. The au
thors wish to thank Dr. A. A. Axelrad for providing one of the
cell lines used in the investigation.
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