[CANCER RESEARCH 36, 1084-1093, March 1976]
Surface Localization of Virus Production on a Glucocorticoid
stimulated Oncornavirus-producing Mouse Mammary Tumor
Cell Line by Scanning Electron Microscopy'
Mafthew A. Gonda,2 Larry 0. Arthur, Victor H. Zeve, Donald L Fine, and Kunio Nagashima
FrederickCancerResearchCenter(Litton Bionetics,Inc.),Frederick21701jM. A. G.,L. 0. A., D. L. F.,K. N.),and the NationalCancerInstitute,NIH,USPHS,
United States Department
of Health, Education
and Welfare, Bethesda,
Maryland
SUMMARY
A chronically infected continuous mouse mammary tu
mon cell line containing virus particles of type B morphol
ogy, free of contaminating type C vinions, has been grown in
tissue culture. These cells were treated with dexametha
sone, a synthetic glucocorticoid, a potent stimulator of
mouse mammary tumor virus expression. Surfaces of un
treated and dexamethasone-treated cells were investigated
by scanning
electron
microscopy.
Untreated
cells
demon
strated a moderate expression of mouse mammary tumor
virus (80 particles/cell) distributed diffusely over the cell
surface. However, vinions on dexamethasone-treated cells
were localized in clusters of 100 to greater than 2000 virus
particles,
often with more than one cluster
20014 (V. H. Z.J
peanance and distribution of diverse types of budding virus
on the cell surfaces (8, 11, 19, 24, 26).
We examined by SEM and TEM a continuous cell line,
Mm5mt/c,, derived from a C3H/Cgnl mouse previously de
scnibed (17) and shown to be chronically infected with a
type B MMTV, free of contaminating type C vinions (6, 7, 17).
Furthermore, treatment of Mm5mt/c1 cells with a synthetic
glucocorticoid, DXM, has been demonstrated to stimulate a
10- to 20-fold increase of MMTV expression over untreated
cells (6, 20). In this conrelat'rve study, the effect of DXM on
the ultrastructural characteristics of the continuous cell line
Mm5mt/c1 and the production, localization, and surface
architecture of budding MMTV during DXM stimulation are
described.
pen cell. Dexa
methasone-treated cells typically showed a 10-fold increase
in cell-associated virus over untreated cells. Concentrated
extracellular fluids from untreated and dexamethasone
treated cultu neswere quantitated for free virus. Dexametha
sone-treated culture fluids demonstrated a similar 10-fold
increase of extracellulan particles, in contrast to untreated
cultures. This increase in virus particles on the cell surfaces
as well as in the extracellulan fluids supports the theory that
dexamethasone has a stimulatony effect on viral replication,
not just on the release of budding particles. The ultrastruc
MATERIALS AND METHODS
Cell Cultures. The C3H/Cgrl mouse mammary tumor cell
line, Mm5mt/c,, originally obtained from Dr. A. Hackett,
Naval Biomedical Research Laboratories, Oakland, Calif.,
was maintained on Dulbecco's modified Eagle's medium
(high glucose) containing 10% heat-inactivated fetal calf
serum, insulin (10 @g/ml),tylocine (60 @g/ml),penicillin
(100 units/mI), and streptomycin (100 pg/mI). For glucocor
ticoid-stimulated cultures, DXM (Sigma Chemical Co., St.
tune of budding
mouse mammary
tumor virus during dexa
Louis, Mo.) was included at a concentration of 10@mole, as
methasone stimulation, determined by scanning and trans
previously described (6, 7). The cells were negative for
mission electron microscopy, and the significance of such
an in vitro system for viral immunodiagnosis are discussed.
mycoplasma by bioassay (Flow Laboratories, Rockville,
Md.) and by the [3Hlthymidine-incorporation method of To
daro et al. (25).
INTRODUCTION
Electron Microscopy. Cells for electron microscopy were
seeded at densities of 4 x lOSin 60-mm carbon-coated Petni
The SEM3 has recently been applied to studies on the dishes (Falcon Plastics, Los Angeles, Calif.) containing 4
surface features of normal and tumor cells in culture (10, 18, carbon-coated 5-mm square glass covenslips, and 5 ml of
19, 21, 22). The 3-dimensional image, high resolution (50 to the growth medium with or without DXM. Cells were grown
100 A), and access to large numbers of whole cells in situ to confluency (4 to 5 days) with no medium changes in a
have made the SEM amenable to examining virus-induced
humidified 10% CO@atmosphere at 37°.Several controlled
cell surface alterations (11, 18, 19, 22, 26). In particular, the experiments were performed, with consistent results.
SEM has been utilized effectively to demonstrate the ap
Prior to fixation, the fluid supernatants from triplicate
untreated and DXM-treated cultures were removed to just
above the cell surface. Five ml of 37°serum-free medium
23294 with the Virus-Cancer Program of the National Cancer Institute.
were added, agitated, and then removed from the Petnidish
? To whom
requests
for reprints
should
be addressed.
3Theabbreviations
usedare:SEM,
scanning
electron
microscopy;
TEM. to dislodge and collect any possible adsorbed virus parti
transmission electron microscopy; MMTV, mouse mammary tumor virus;
des. The original culture medium and serum-free wash
DxM,dexamethasone.
Received September 19, 1975; accepted November 13, 1975.
from each dish were pooled and placed in individual centni
,This
investigation
was
supported
byUSPHS
NO1-CO-25423
and
NO1-CN
1084
CANCER RESEARCH VOL.36
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Cell Surface Localization of MMTV by SEM
fuge tubes for concentration and for subsequent virus parti
cle quantitation.
Cells selected for electron microscopy were fixed at 37°
for 30 mm in 2% glutaraldehyde in 0.5x Dulbecco's phos
phate-buffened saline and 0.05 M cacodylate buffer, pH 7.2,
and then postfixed for 5 mm with 1% osmium in 0.2 M
cacodylate buffer. The osmium was quickly replaced by
cacodylate buffer, and the covenslips were placed in a cniti
cal point drying specimen holder submerged in cacodylate
buffer. Cane was taken to avoid air drying. The remaining
fixedcellsin Petnidisheswere refrigerated
at1@Y@
forlater
dehydration and for in situ embedding for TEM. Cells for
SEM were rapidly dehydrated in graded ethanols with 3 final
changes of 100% ethanol. Specimens were preserved
against air-drying artifacts by critical point drying with
Freon, according to the method of Cohen et al. (3), using a
Boman SPC-EX-900 device (The Boman Co., Tacoma,
Wash.). After critical point drying, the specimens were at
tached to 13-mm aluminum stubs with silver conducting
paint and were lightly carbon coated in a vacuum evapora
ton. The specimen stubs were then removed, placed in a
Conductavac I diode sputtering device (Seevac Corp., Mon
roeville, Pa.). They were then coated at a pressure of 175
@m,
75 ma, at a distance of 4.5 cm from the cathode source
for 6 mm with 75 to 100 A of gold-palladium to facilitate
visualization of cell surfaces by minimizing surface charg
ing (10).
Specimens were photographed in an Etec Autoscan open
ated at 17 kV and equipped with a 90°goniometer tilt stage.
Micrographs were taken at a 35°specimen tilt.
For TEM correlation, cells from triplicate cultures previ
ously mentioned for in situ embedding in Petni dishes were
poststained in a sucrose-buffered 0.25% unanyl acetate so
lution for 2 hn, dehydrated in graded ethanols, and infil
trated with 100% Epon 812 at 25°for 2 days to allow the
residual ethanol to volatilize. The infiltrated specimens were
then polymenized at 50°for 24 hn, followed by 70°for 48 hr.
Thin sections were cut perpendicular to the growth plane of
cells on a LKB Ultrotome
Ill equipped
with a diamond
knife,
mounted on carbon-coated Fonmvan 300 mesh copper
grids, and double stained with a saturated solution of uranyl
acetate and Reynolds lead citrate. Observations and micro
graphs were made with a Hitachi HU-12A TEM operated at
75 kV.
Virus Particle Quantitation. For extracellular virus parti
cle quantitation, the pooled fluids previously isolated were
centrifuged at 100,000 x g for9O mm. The clarified superna
tant fluid was removed, and the remaining virus pellet was
resuspended in 100 pi of 0.5x Dulbecco's phosphate
buffered saline, pH 6.5, by rigorous agitation. The 50-fold
concentrated virus was counted via an automated particle
analysis system interfaced to a SEM, by methods previously
described by Zeve et al. (28).
Cell surface-associated virus was quantitated manually
with a visual method by randomly selecting 10 cells from
micrognaphs taken of each of the above-mentioned tnipli
cate untreated and DXM-tneated cultures. Each cell was
scored for numbers of virus particles, and an average of the
number of virus particles/cell for each dish was calculated.
RESULTS
Surface Distribution of MMTV on Mm5mt/c Cells. Con
fluent untreated cultures of Mm5mt/c1 cells by SEM are
depicted in Fig. 1. The cells were flattened and epithelioid in
appearance. Contact between contiguous cells was made
by an overlapping of thin, flat lamelloplasm. Surface projec
tions, budding virus either sessile from the cell membrane
or from microvilli, moderately covered the cell surface and
were most sparse oven the nuclear region. Individual virus
particles had an approximate diameter of 0.15 @tm.
A higher
magnification of the surface ofa cell in the centenof Fig. 1 is
shown in Fig. 3. With the exception of cell-associated virus,
the surfaces were relatively smooth.
Confluent DXM-tneated Mm5mt/c1 cells (Fig. 2) were simi
Ian to the untreated cells (Fig. 1) in their flattened and
epithelioid appearance; however, DXM-treated cells dif
fered dramatically in both the appearance and relative num
ben of cell-surface projections. Massive, clustered areas of
budding virus appeared on the cell surface, usually away
from the nuclear region and oven areas of thin, flat cyto
plasm. Virus particles of the same diameter as those in
untreated cells budded from microvilli, surface folds, and
sessile from the cell membrane. Clusters of virus in compact
formation contained as many as 100 to more than 2000
particles, with sometimes more than 1 cluster appearing on
the surface of a DXM-treated cell. Some diffuse distribution
of budding virus also occurred. Fig. 4 shows a higher mag
nification of localized MMTV production from a marginal
area of a cell in the center-left portion of Fig. 2, which
reveals the uniform appearance of the budding virus.
TEM Correlation of SEM Surface Features. Although
Mm5mt/c1 cells are chronically infected with MMTV, un
treated cells revealed an occasional virus budding from the
cell surface and microvilli by TEM. Most often, TEM of thin
sections of untreated cells demonstrated the surfaces (Fig.
5) to be devoid of numerous surface projections, which is
not surprising since it correlates well with the moderate
surface activity seen in SEM of untreated cells (Figs. 1 and
3).
In agreement with the impressive number of budding
vinions by SEM (Figs. 2 and 4), TEM examination of DXM
treated cells revealed many budding vinions, most fre
quently in areas of thin, flat cytoplasm away from the nu
clear region (Fig. 6). The detail of budding MMTV still asso
ciated with the surface membrane in Fig. 6 is more apparent
in Fig. 7 (see also Fig. 14). MMTV's are essentially round
with an approximate diameter of 0.12 @m
and have a cen
trally located doughnut-shaped nucleoid of 2 concentric
rings with an electron-lucent center, a distinct intermediate
layer, and an outer spiked envelope (4, 5, 23). The small size
differences between TEM measurements of vinions (0.12
@m)and SEM measurements (0.15 @.tm)are due to the
presence of a conductive metal coating applied to the SEM
cells to facilitate their visualization, as described in “Mateni
als and Methods.―
Occasionally, desmosomes (not shown) were present in
areas of contact between contiguous cells in thin sections
of both untreated and DXM-treated Mm5mt/c, cultures, yen
ifying the origin of these cells as epithelial (10, 17).
MARCH 1976
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1085
M. A. Gonda et al.
Ultrastructure of MMTV on DXM-treated Cells. With
DXM-tneated Mm5mt/c, cells visualized by the SEM, we
observed
MMTV
budding
from
micnovilli,
surface
folds
(evaginations), and sessile from the cell membrane. The
nature
of these
virus-cell
associations
is more
apparent
in
Figs.8 to 11.
Fig. 8 shows the marginal edge of a DXM-treated Mm5mt/
CI
cell.
Single
round
sessile
particles
in
various
stages
as bead-like
structures
(hereafter
of cell-associated
Table 1
and extracellular
virus in untreated
and DXM-treatedMm5mt/c1cultures
The t test was applied
for cell-associated
virus (p < 0.01) and
extracellular virus (p = 0.014)to show significant difference be
tween treated and untreated
cultures.
Cell-associated
vi rus
(viruses/cell)
of
budding can be seen protruding from the surface mem
brane. Sessile budding particles also occurred in multiples
(duplex, triplex, etc.). Other vinions (Fig. 8), still associated
with each other and confined within the same intact mem
brane, appeared
Comparison
0.14DXM-treated
Untreated culture80.7
culture1036
Extracellular vi
rus (viruses/mI
culture)
± 10.3k108.11±
±216.61@'@±
0.14
a Mean ±SE.
called
moniliform) which bore a nesemblence to a multiconstnicted
at their surfaces on released them into the extracellular
fluids, 50-fold concentrations of the culture fluids from the
containing vinions projected from the cell surface. Obsenva above-described triplicate cultures were quantitated and
tions of many such moniliform structures by SEM sug
compared in Table 1.
gested that several shared a common origin on the cell
These data show that there was a 10-fold increase in the
surface from which the virus is expressed. A similar mar
number of cell-associated MMTV on the surfaces of DXM
ginalarea of a treatedcellisshown in Fig.9. Budding treated cells, with a similar 10-fold increase in the number of
MMTV occurred singly on the surface, on as monilifonm free MMTV occurring in the extracellulan fluids of DXM
structures. Virus budding from micnovilli and from surface treated cultures.
folds can also be observed. At higher mangifications (Figs.
The data inTable 1 were subjectedto the t test,
which
10 and 11),thepinchingoffofmaturingvirions
from micro showed a significant difference between treated and un
villi and from surface folds is discernible. Virus appeared to treated cultures for both cell-associated and extracellular
bud readily, not only from the tips of microvilli, but margin
virus. This evidence further supports the theory that DXM
ally, lateral to the long axis of microvilli.
stimulates enhanced virus replication, not just a release of
TEM correlation of DXM-treated cultures revealed many virus particles in the process of budding.
microvillus
virus-cell
in size and shape. These moniliform
associations
that closely resembled
structures
those seen by
SEM. Moniliform processes (Figs. 12 and 13) were fre
quently seen in thin sections. These processes sometimes
demonstrated aberrant nucleoids of a cylindrical shape.
Atypical forms of budding virus were not seen in untreated
cells. This finding suggests that while DXM increased total
virus production (6, 7, 20, 27) it also stimulated aberrations
in virus
assembly
and maturation.
Similar
atypical
particles
have been noted in other in vitro systems in which virus
stimulating
drugs were used (16).
Single, sessile particles budded from flat areas of cell
surfaces (Figs. 6, 7, and 14) and from raised surface folds
(Fig. 14). Multiple budding from microvilli both terminally
and laterally (Fig. 14) was still another form of virus expres
sion in DXM-tneated cells. Although not shown in any of the
micrographs, virus was found to bud on the ventral side
(side associated with substrate) of both untreated and DXM
treated Mm5mt/c1 cells. This form of budding was moderate
in comparison
to the dorsal
cell surface
and seemed
to be
confined to intercellular spaces; however, its contribution
to the quantity of extracellulan virus cannot be excluded.
TEM evidence, from both morphological stages of matuna
tion (4) and the continuity of the membrane of the viral
nucleoid with the cell membrane, confirms that the virus
particles seen by the SEM have not been readsorbed after
budding but are actually in the process of budding.
Quantitation of Surface and Extracellular Virus. Surfaces
of cells were analyzed for numbers of cell-associated virus
particles pen cell from triplicate cultures of untreated and
DXM-treated Mm5mt/c1 cells. The results of these findings
are shown in Table 1. To ascertain further whether DXM
treated cells merely retained the increased number of virus
1086
DISCUSSION
This communication describes ultrastructu nal character
istics by SEM and TEM of a continuous mouse mammary
tumor cell line, Mm5mt/c1, grown in tissue culture with and
without DXM stimulation. Untreated cell surfaces by SEM
typically showed moderate surface budding with a diffuse
distribution of viral particles. SEM studies of the budding
process of unstimulated cells infected with type C munine
onconnavinuses have shown a similar random distribution
(19, 26). In contrast, the most striking feature of the DXM
treated Mm5mt/c1 cell surfaces, lacking on untreated cells,
was the impressive increase in MMTV localized in massive
compact clusters.
A recent SEM study by Wong and MacLeod (26) sug
gested that up to 10% of the infected cell surface may be
covered by type C viruses in the process of budding. In this
study, MMTV on untreated cells occupied considerably less
surface area than that reported for type-C virus. In contrast,
MMTV on DXM-treated
cells occupied
as much as 30% of
the cell surface (unpublished data), with sometimes more
than 2000 MMTV particles per cell.
The localization of virus on DXM-treated cells appeared to
be in areas of thin, flat cytoplasm away from the nuclear
area. Localized areas of MMTV expression may reflect dif
ferences in the cell membrane over the peripheral cyto
plasm versus the nuclear region when Mm5mt/c1 cells are
grown in monolayer.
These unique areas of the cell surface visualized by SEM
during DXM stimulation demonstrated various budding
CANCER RESEARCH VOL.36
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Cell Surface Localization of MMTV by SEM
processes. The most common method of virus maturation
was for virus to bud as single on multiple sessile particles
from the plasma membrane. Other vinions budded from the
tips on marginal areas of microvilli. The budding of MMTV
from the plasma membrane and from microvilli has been
shown (1, 5, 12, 13).
A frequent phenomenon, to our knowledge not previously
described for virus of type B morphology, was the expnes
sion of maturing virus from the cell surface as moniliform
processes which contained as many as 100 viral nucleojds.
The fact that several of these moniliform processes shared
a common origin on the cell surface further suggests differ
ences in the cell membrane in areas of localized virus ex
pression. Whether these processes are released intact, in
portions, or as single particles has not been determined.
SEM (11) and TEM surface replica (2) studies of the budding
of vesicular stomatitis virus from unstimulated cells have
demonstrated microvilli processes from which vinions bud.
The budding of virus from surface folds was another form
of viral maturation observed in DXM-treated cells that has
not been reported previously. Correlative ultnastructunal
studies by SEM and TEM of virus-infected cells grown in
monolayer offer the distinct advantage of verifying the exist
ence of such unique areas of viral expression that could not
be obtained with certainty from either method alone.
DXM-treated cell surfaces demonstrated a 10-fold in
crease in budding MMTV. TEM of untreated and DXM
treated Mm5mt/c, cells correlated the identity of budding
MMTV and further verified the enhanced expression of
MMTV on DXM-treated cells seen by the SEM. A similar 10-
fold increase in extracellular virus was also found in con
centrated supernatants of DXM-treated cultures. Although
the exact mechanism of action of DXM on increasing MMTV
expression is unknown, recent studies (20) have shown an
increase of viral RNA and MMTV antigen, subsequent to
DXM treatment. These studies suggest an increased tran
scniption of the integrated viral genome.
The present studies support the contention that DXM acts
to enhance production of MMTV in vitro. The distribution of
viral particles appears to be localized in specific regions of
the cell surface which may represent sites of viral antigen
expression. In addition to the enhanced production of
MMTV, preliminary
studies
on DXM-stimulated
cells pro
ducing Mason-Phizen monkey virus and C-type munine on
cornaviruses suggest a stimulatony effect of glucocorticoids
on these viruses.
From a practical standpoint, the SEM technology demon
strated here may represent a method of screening for virus
producing cell lines by examining cell surfaces for budding
particles, rather than the extracellular supennatants for free
vinions. The high resolution of SEM, coupled with its ability
to readily display the surfaces of large numbers of whole
cells in situ, further permits approaching such problems as
viral antigen localization by immunological cell surface Ia
beling with visual markers for the SEM (9, 14, 15). The
Mm5mt/c1 cell line presented here offers a novel in vitro
approach to immunological cell surface mapping studies
for antigenic sites, using recognizable visual markers. Im
munodiagnostic studies of this nature, as well as studies on
the stimulatony effects of DXM on other virus-producing
cell
lines, are now being performed in our laboratory.
ACKNOWLEDGMENTS
The authors wish to acknowledge the excellent technical assistance of
Merle Hartsock for cell cultures, Sally Sevy for photography, Charles Riggs
for statistical analysis, and Amy Huter for editing and typing of this manu
script.
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Figs. 1 to 14. Representative SEM and TEM micrographs from untreated and DxM-treated Mm5mt/c, cells.
Fig. 1. SEM micrograph of untreated cells at confluency. Surfaces are sparsely covered by budding MMTV (arrows) generally away from the nuclear area
(N). x 5,000.
Fig. 2. SEM micrograph of DxM-treated cells at a density similar to those shown in Fig. 1. Massive clustered areas of budding MMTV(V) as well as diffusely
distributed budding MMTV are apparent. The nuclear regions (N) are also shown. x 5,000.
Fig. 3. SEM micrograph at higher magnification of a portion of the surface of a cell in the center of Fig. 1. With the exception of budding MMTV, the
surfaces are relatively smooth, x 11000.
Fig. 4. SEM micrograph which shows localized MMTV production and uniform round appearance of budding-virus from a marginal area of cell in the
center-left portion of Fig. 2 at a higher magnification. x 11,000.
Fig. 5. TEM micrograph of a thin section of untreated cells. Surfaces are devoid of numerous surface projections, which correlates well with the moderate
surface activity of untreated cells shown in Figs. 1 and 3. x 10,000.
Fig. 6. TEM micrograph of thin sections of DXM-treated cells. The surface reveals many budding MMTV, most frequently from an area of thin, flat cytoplasm
away from the nuclear region (N). x 9,000.
Fig. 7. An area of budding MMTV from Fig. 6 showing typical type B morphology of virus at a higher magnification. x 37,500.
Fig. 8. SEM micrograph of cell margin of DXM-treated cell. Single sessile virus(Vs), moniliform processes containing viral nucleoids(Vm), and the origin of
several moniliform processes (OVm) from which virus is expressed are visible. x 17,850.
Fig. 9. SEM micrograph of an area of localized virus production on DXM-treated cells similar to that shown in Fig. 8. Single and multiple-budding sessile
virions, moniliform processes, and virions budding from surface folds (F) are demonstrated. x 17,850.
Fig. 10. Surface of a DXM-treated cell. The pinching off of maturing virus from microvilli and surfacefolds is discernible(Vb). Virus appeared to readily bud,
not only from the tips of microvilli, but marginally,lateral to the long axisof microvilli, by SEM.x 39,250.
Fig. 11. SEM micrograph of another area of localized virus expression of a DxM-treated cell showing virus budding (Vb) lateral to the long axis of rnicrovilli.
Many single and multiple sessile virions are also apparent. x 42,850.
Fig. 12. TEM micrograph of a moniliform process containing viral nucleoids produced from a DXM-treated cell. Aberrant nucleoids in these structures were
sometimes seen. x 62,500.
Fig. 13. TEM micrograph of another moniliform process from a DXM-stimulated cell. Normal nucleoids were encountered in this thin section. x 81,250.
Fig. 14. TEM micrograph of a DXM-treated cell showing MMTV budding sessile from the surface membrane, surface folds (F), and lateral from microvilli
(M). x 54,250.
1088
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1093
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research.
Surface Localization of Virus Production on a
Glucocorticoidstimulated Oncornavirus-producing Mouse
Mammary Tumor Cell Line by Scanning Electron Microscopy
Matthew A. Gonda, Larry O. Arthur, Victor H. Zeve, et al.
Cancer Res 1976;36:1084-1093.
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