Replication of mouse mammary tumor virus in tissue culture

VIROLOGY

77.

12x30

Replication I. Establishment

NURUL Memorial

(1977i

of Mouse Mammary

Tumor Virus in Tissue Culture

of a Mouse Mammary Tumor Cell Line, Virus Characterization, Quantitation of Virus Production

H. SARKAR,’

Sloan-Kettering

Cancer

ANTHONY Center,

Neuj Camden.

A. POMENTI, York, New! York 10021, New Jersey 08103

Accepted

October

AND

ARNOLD

and Institute

for

and

S. DION Medical

Research,

6,1976

The establishment of an epithelial cell line (MuMT-73) derived from spontaneous mammary tumors of BALBlcfC3H mice as a source for the continuous production of mouse mammary tumor virus (MuMTV or B particles) is described. Morphological, immunological, and biochemical techniques were used to characterize the virus. It was found that, under conditions of normal cellular growth, the cells produced only type-B particles; no concomitant synthesis of type-C particles was detected. A unique feature of this cell line is that the cells do not produce detectable amounts of intracytoplasmic typeA particles, but many B particles were found to assemble at the cell surface with gradual development of the nucleocapsid. The kinetics of virus release into tissue culture medium after transfer of cells was studied quantitatively by particle counting and viral reverse transcriptase (RDDP) measurements. When the medium was changed every 24 hr, the cells continuously produced virus at a slow rate for up to 6 to 8 days and thereafter at an increased rate for up to 12 days. After this period, virus production gradually declined but did not decrease to the basal level. During the period of high level virus production, about 30-4096 of the cells were found to produce MuMTV antigens. By estimating the total number of virus particles in the culture medium and by counting the number of antigen-positive cells, it was found that over a 20-hr period during optimum virus production a cell would release approximately 800 virus particles. Treatment of the cells with either hydrocortisone or dexamethasone resulted in an increased production of type-B particles. An additional increase in virus production was obtained when the cells were treated with insulin together with either hydrocortisone or dexamethasone.

tempts have been made by various investigators to establish cell lines from spontaneous mammary tumors in order to provide a convenient source of MuMTV. Some of these cell lines produced only low amounts of MdKI’V (Sykes et al., 1968; Lasfargues et al., 1972; Owens and Hackett, 1972), while in other lines spontaneous production of MuMTV and/or murine leukemia virus (MuLV) was observed (Dmochowski, et al., 1971; Hilgers et al., 1972; Shigematsu et al., 19711. To study the kinetics of MuMTV replication and cell-virus relationships and to understand viral morphogenesis, it is necessary to have cell lines which produce sufficient amounts of MuMTV without detectable amounts of

INTRODUCTION

Investigations into the biophysical, biochemical, and immunological properties of mouse mammary tumor virus (MuMTV) have been carried out mainly with virus purified from the milk of mice with a high incidence of breast cancer (Nandi and McGrath, 1973). Short-term cultivation of mammary tumor cells in vitro has also provided a suitable system for harvesting virus and for studying the kinetics of virus replication (Cardiff et al., 1968; McGrath, 1971; McGrath et al., 1972). Several atI Address reprint requests kar, Sloan-Kettering Cancer enue, New York, New York

to Dr. Center, 10021.

Nurul H. 1275 York

SarAv12

Copyright All rights

0 1977 by Academic Press, Inc. of reproduction m any form reserved.

ISSN

0042-6822

13

REPLICATION OF MuMTV other viruses. The cell lines established by Sykes et al. (1968)) Lasfargues et al. (19721, and Owens and Hackett (1972) do not produce significant quantities of MuLV but release only low amounts of MuMTV particles. The yield of MuMTV from the Sykes’ cell line (CLL-51) can be increased by passing the cells of this line through newborn mi.ce of the C57BL, Af, and A strains; however, such procedures also result in the simultaneous production of MuLV (Lasfargues et al., 1970). About 4 years ago, we started culturing mouse mammary tumor cells in vitro with the goal of establishing a cell line which would produce large quantities of virus. After several efforts with mammary tumors from various strains of mice (RIB, GR, and A), we used cells derived from a combination of three spontaneous mammary tumors from BALB/cfC3H mice and were able tc establish a cell line (MuMT73)’ producing large amounts of virus. Meanwhile. there have also been several encouraging reports about tissue culture sources of virus. Parks and Scolnick (1973) have been able to establish a high virus producing clonal line (clone 11) from the Sykes’ Cl151 line; Yagi (1973) has described a cell line (MJY-alpha, derived from BALBlcfC3H mammary tumor) that also produces a large quantity of virus. Furthermore, methods have been devised to enhance the replication and release of MuMTV in various cell lines by treating them with dexamethasone, a synthetic glucocorticoid hormone (Parks et al., 197413, 1975; Fine et al., 1974; Dickson et al., 1974; Ringold et al., 1975). The present report describes certain aspects of MuMTV morphology, the kinetics of virus production in the MuMT-73 cell line in a quantitative manner, and the characteristic biochemical and immunological properties of the tissue (culture-grown virus. MATERIALS AND METHODS (a) Cell Culture Tumor cells were obtained from spontaneous mammary adenocarcinomas which 2 The symbol MuMT represents Murine mary Tumor and the fact that the cell line originated in 1973.

Mamwas

had developed in RIII, GR, A, or BALB/ cfC3H mice. Individual tumors from RIII, GR, or A strain mice or a pool of three tumors from BALB/cfC3H mice were used. Tumor cells were dissociated from the minced tumors by 0.25% trypsin in Ca2+and Mg2+- free Hanks’ balanced saline solution (BSS). Cellular debris and large aggregates of cells were allowed to sediment for several minutes under gravity. The supernatant was then centrifuged at 300 g for 10 min. The resulting supernatant was then discarded, and the cell pellet was resuspended in basic growth medium (Hanks-Eagle’s minimum essential medium) supplemented with 15% fetal calf serum (Flow Laboratories, Rockville, Maryland), 10 pglml of insulin, 100 U/ml of penicillin and 100 Fg/ml streptomycin. Cells were plated at a density of 10’ cells/ flask (25-cm2 flask; Falcon Plastics). The first subculture of the primary cells was done into 75-cm” flasks 4 weeks after plating. After 13 weeks, the cells were passed every 7 days in a 1:4 ratio, and the medium changed twice a week. When virus was to be harvested, the cells were usually seeded at a concentration of 2-4 x 10” cells/75 cm’ and maintained in the medium described above to which hydrocortisone (10 pg/ml) had been added. Since several studies have shown that glucocorticoid hormones enhance the production of MuMTV in cultured murine mammary tumor cells (McGrath, 1971; Parks et al., 1974b, 1975; Fine et al., 1974; Dickson et al., 1974; Ringold et al., 1975; Young et al., 1975), preliminary experiments were performed to establish the effect of insulin, hydrocortisone, and dexamethasone on virus replication in MuMT-73 cells. (b) RadiolabeLing

of Virus

Virus was radiolabeled by adding radioactive precursors to the medium of virusproducing cells in vitro. Cells were incubated for 24-72 hr in the presence of labeled precursors before harvesting the ‘virus-containing medium. The quantities of radioactive precursors used were: 10,15, or 20 $Zi of ‘%-labeled L-amino acid mixture/ml, 10 &i of [:‘Hlglucosamine/ml, or 20 &i of [:
14

SARKAR,

Nuclear setts.

Corporation,

(c) Isolation

Boston,

POMENTI,

Massachu-

of Virus

Tissue culture medium was first centrifuged at 5000 rpm for 10 min in a Beckman SW 27 rotor to remove the cells and cell debris. The resulting supernatant was centrifuged at 25,000 rpm for 90 min in the same rotor. The pellet was soaked overnight in 100 ~1 of phosphate-buffered saline (PBS), pH 7.4. The resuspended pellet was layered on a preformed continuous sucrose gradient (O-60%, w/v) and centrifuged at 30,000 rpm for 2 hr in a Beckman SW 50.1 rotor. Visible virus bands or radioactive peaks at a density of 1.17-1.18 gl cm{ were collected, diluted with PBS, and recentrifuged as above for 1 hr. The final virus pellet was resuspended in 100 ~1 of PBS. (d)

Electron

Microscopy

(i) Thin sections. Cultured cells were scraped from the flask with a rubber policeman, washed twice in PBS, fixed in 2.5% gluteraldehyde for 1 hr, postfixed in 1% osmium tetroxide, dehydrated in graded alcohols, and embedded in Epon812 (Luft, 1961). Thin sections were cut with an LKB ultramicrotome and sequentially stained with uranyl acetate and lead citrate according to the methods of Reynolds (1963). (ii) Negative staining. A drop of purified virus suspension was placed on carbon-coated microscopic grids; after 1 min the excess fluid was withdrawn, and immediately a drop of 2% sodium phosphotungstate (PTA), pH 7.0, was put on the grid. The virus was stained for lo-15 set and air dried. Purified virus was also examined after glutaraldehyde (2.5%) fixation (5 min) on grids followed by PTA staining. (iii) Counting of virus particles. Quantitative particle counting was made following the method described by Watson et al., (1963). Pellets of purified virus obtained from a known volume of tissue culture fluid or virus concentrated by high speed centrifugation (25,000 rpm in an SW 27 rotor for 90 min) of culture fluids, prespun

AND

DION

at 5000 rpm for 10 min (Beckman SW 27 rotor), were resuspended in 100 ~1 of PBS. An equal volume of latex particle suspension was added to the viral preparations and thoroughly mixed. The number of latex particles per milliliter was estimated from the dry weight of a known volume of the suspension (prespun at 5000 rpm for 10 min in an SW 39 rotor to eliminate latex aggregates), the density, and the diameter of the particles. Dilution of the original latex suspension was made in PBS to give a concentration of 4.7 X lo” particles/ml. A drop of the mixture was placed on a carbon-coated microscopic grid; after 1 min the excess fluid was withdrawn with a piece of filter paper and immediately stained with PTA. We designate this technique a drop method. The number of virus particles per milliliter in tissue culture fluid was estimated from the ratio of the number of virions to the number of latex particles observed in a randomly selected field on the screen of the microscope. The reliability of the technique was established by using a highly purified preparation of virus obtained from RI11 mouse milk. In these experiments, random dilution of virus was made, the amount of protein was estimated by the method of Lowry et al. (1951), and particle counting was done on a virus-latex mixture sprayed in the presence of PTA (spray method, Watson et al., 1963). The same viral samples were simultaneously prepared by the drop method. There was a good correlation between the amount of protein and the number of virus particles counted by both the spray and the drop method; therefore, all subsequent virus counting was performed by the drop method. (e) Immunodiffusion

For the detection of viral antigens in the high speed pellet of the culture fluid or purified tissue culture virus, ether-treated samples were tested against rabbit antiMuMTV or -MuLV serum in agar diffusion plates (Hyland Laboratories) and left for 24 hr in a humidifed chamber at room temperature. Anti-MuMTV serum was made in rabbits using ether-disrupted virus (Nowinski et al, 1972) purified from

REPLICATION

RI11 milk; antiserum was absorbed with two volumes of virus-free C57BL milk in vitro and then in viva by injecting it into the same strain of mice (Sarkar and Dion, 1975). Cfl Immunofluorescence An indirect membrane immunofluorescence test was performed on trypsinized or scraped viable cell suspensions with rabbit anti-MuMTV or -MuLV serum as described by Klein et al. (1967). The percentage of cells carrying MuMTV antigen on the surface was scored by fluorescence microscopy. Intracellular antigens were studied on acetone-fixed cells grown in monolayers on glass coverslips or glass slides containing preformed wells made by Fluoroglide coating (Hilgers et al., 1972). (g) X-C Test Tissue culture virus was tested by the standard TJV-XC test (Klement et al., 1969; Rowe et al., 1970) using IIIGA cells which are sensitive to both N- and B-tropic murine leu.kemia viruses (Hartley et al., 1970). The test was kindly performed by Dr. T. Pincus. (h) RNA-Directed (RDDP) Assays

DNA

Polymerase

Tissue culture fluids were spun at 5000 rpm for 20 min and then at 9000 rpm for 15 min in a Sorvall fixed angle rotor. The resulting supernatant was layered on an 8ml, 20% glycerol pad and centrifuged at 25,000 rpm for 90 min in a Beckman SW 27 rotor. The viral pellets were resuspended in 100 ~1 of 0.01 M Tris-HCl buffer (pH 8.3) and st.ored at -20”. Twenty-microliter aliquots of the virus suspension were assayed in a final volume of 50 ~1 containing: 25 kg of bovine serum albumin, 3 kmol of dithiothreitol, 0.33% NP-40, 2 pg of poly(rC).oligo(dG) (Collaborative Research, Waltham, Massachusetts), 2.5 pg of actinornycin D, 0.75 ymol of NaF, 5 pmol of ATP, 3.325 pmol of Tris-HCl (pH 8.31, 0.62,5 pmol of NaCl, 0.4 pmol of MgCIZ, and 1.27 pmol of [“H]dGTP (4600 cpmlpmol, AmershamlSearle). After 30 min at 37”, the reaction was terminated by chilling on ice and by the addition of 100 ~1

OF

15

MuMTV

of carrier yeast RNA (300 *g/ml) and 100 ~1 of trichloroacetic acid (TCA) mix (equal volumes of 100% TCA, saturated tetrasodium pyrophosphate, and saturated trisodium phosphate). After filtration, samples were counted in BBOT/toluene in a Packard Tri-Carb scintillation counter. (i) Polyacrylamide-Gel

Electrophoresis

Sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (SDSPAGE) was performed by the method of Shapiro et al. (1971). Fifty microliters of 0.01 M sodium phosphate (PH. 7.8) containing 1% SDS and 1% P-mercaptoethano1 were added to a pellet of purified virus and denatured by heating at 60” for 30 min; glycerol was added to a concentration of 10%. The gels contained 7.5% acrylamide, 0.25% bisacrylamide, 0.1% SDS, and 0.1 M sodium phosphate (pH 7.8). Electrophoresis was performed at 3 mA/gel for 30 min, followed by 3.5 hr at 7 mA/gel. Gels were sectioned at 1.2-mm intervals, digested overnight in 0.2 ml of 30% H,O, at 60”, cooled, and counted in Aquasol in a Packard Tri-Carb liquid scintillation counter. Molecular weights of the viral polypeptides were estimated by comparing their relative migrations with respect to standard proteins. RESULTS

Cell Culture Initially, the cells grew very slowly, and some cells formed discrete epithelioid colonies which enlarged to form a few threedimensional cellular mounds that have been described as “domes” (McGrath et al., 1971). After 4 weeks, the first passage was made from small flasks (25 cm”) to large flasks (75 cm’) at a ratio of 1:l and then at a ratio of 1:2 every 2 weeks for the next four passages (8 additional weeks). At the end of 13 weeks (sixth passage), the cells were growing at a faster rate and were passed (1:4) weekly. The cell line thus established primarily contains epithelial cells (Fig. I). Cells plated at a density of about 2 x 10fi tells/75-cm2 flask attained confluency within 4-6 days and could be maintained up to 16-20 days with medium

FIG. 1. Photographs of the mouse mammary tumor cells (MuMT-73) at passage No. 100. Appearance of cells after 1 (a), 10 (b), and 16 Cc) days of transfer. The monolayer cells in tb) and CC) are definitely epithelold in morphology; formation of domes is a characteristic feature of these cells when plated at cell densities greater than 10’ tells/75-cm’ flask. (a), 175x. tbl and CC), 135x. 16

REPLICATION

OF

changes twice per week. Cells cultured routinely in this schedule produced only occasional domes. However, cells plated at higher density (10’ cells or mare/75-cm” flask) always produced domes of varying sizes (Fig. 1). The growth characteristics of the cells are illustrated in Fig. 2. Virus

Production

At the early stages of cell culture, the production of virus was monitored only by negative staining of the high speed pellet fraction of the culture medium. When the cells were {growing at a faster rate, thin sectioning of the cells was done to observe the assembly of the virus at the cell surface. Throughout the different stages of cell growth, quantitative release of virus into the culture was assayed by particle counting and by measuring the amount of RDDP activity associated with the particles; accumulation of virus-specific antigen at the cell surface was determined by immunofluorescence. (a) Electron microscopy. Thin section electron microscopy of the cultured cells revealed the presence of budding as well as extracellular mature type-B virions (Fig.

I I

~-1~ 6

Days

L--.-1-1IO 8

after

J 12

;4

16

passage

FIG. 2. Growth characteristics of the cultured MuMT-73 cells (passage number 90) after transfer. Confluent cells from two large flasks (75 cm’) were trypsinized, pooled, and resuspended in growth medium. Viable cells, 2 x 1Oj, were plated in each of nine flasks (25 cm’); counting of live cells was made on various d.ays.

MuMTV

17

3). It is interesting to note that many particles were found to bud in a manner similar to the budding of type-C particles (arrows in Fig. 3a; Figs. 3c and d); that is, the nucleocapsid formed a double-shelled structure immediately below the cell membrane at the site of the viral bud. This structure appears as a crescent-like structure. However, the presence of projection(s) or spikes on the membranes of such particles makes it certain that these particles are of B type. These results demonstrated that no preformed A particles were involved in the morphogenesis of these MuMTV particles produced by the MuMT-73 cells. Many particles with complete cores having the morphology of A particles were observed in the process of budding, However, it is doubtful whether preformed A particles were involved in the formation of typical B-type buds, since we did not observe any intracytoplasmic A particles in the approximately 100 different cell sections containing more than 3000 budding particles that we examined. It is possible, however, that in this particular cell system, A particles are formed preferentially beneath the cell membrane. Although the sizes of the virus particles were fairly uniform, a minor portion of the budding (Fig. 3c) and the extracellular mature particles (Fig. 3h) were observed to be large. Mature particles containing two (Fig. 39 or three nucleoids were observed occasionally; usually these particles were large. The particles produced by the MuMT-73 cells were confirmed as type B by the negative-staining technique (Fig. 4) in which the surface projections of the virions were resolved more clearly than in thin sections. (b) Viral antigens. In addition to the morphological identity of the virions as type-B particles, immunological analyses were performed to ensure further that the cultured cells produced only MuMTV particles. Viable cells were used to detect cell membrane-associated viral antigen. Rabbit anti-MuMTV serum produced positive membrane immunofluorescence, but no specific fluorescence was observed when the cells were similarly tested with rabbit anti-MuLV serum. Monolayer cells after fixation with acetone were frequently used

FIG. 3. Thin section electron micrographs of MuMT-73 cells showing various forms of budding and mature particles. Typical bud of MuMTV (tb) with preformed cores and atypical buds tab) with incomplete cores (panel a). Budding and extracellular mature MuMTV (B) are shown in (b). (c), Variation in size of the budding MuMTV (with incomplete cores) particles; the particle on the right is bigger than the other two particles. The projections or spike(s) on the budding particles with incomplete or complete cores are resolved in most of the micrographs. Panels f-i, MuMTV particles of normal size I (f) and tg)l or larger I(h) and (ill, with one nucleoid [ tfi-(h)] or with two nucleoids (i) (a), 41,800~; (b). 62,700~; (c) and (f)-(i), 105,500~; Cd) and te), 123,500x. 18

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OF

MuMTV

19

FIG. 4. Typical MuMTV particles observed by the negative-staining techniques. Particles prefixed with 2.5% gluteraldehyde and stained with PTA (pH 7.0) show nearly spherical morphology (al. MuMTV without prior fixation shows typical head and tail configuration after PTA staining (b). (a), 70,000~; tb), 100.000x. FIG. 5. Cytoplasmic immunofluorescence reaction of the MuMT-73 cells with rabbit anti-MuMTV serum. Note the positive and negative cells.

20

SARKAR.

POMENTI,

in the indirect immunofluorescence test. A certain percentage of the cells was always found to be positive for MuMTV antigens (Fig. 5), but all were negative for MuLV antigens. Occasionally, dome-producing cells were examined similarly; the multilayered dome cells as well as the surrounding epithelial cells in monolayer both contained MuMTV antigens. Double immunodiffusion precipitation analyses were also done with anti-MuMTV and anti-MuLVp30 sera against the density gradient-purified virus harvested from the tissue culture medium. Ether-disrupted Rauscher leukemia virus was used as a positive control. Anti-MuMTV serum reacted with the tissue culture-grown virus and produced two immunoprecipitation lines in immunodiffusion; but the virus did not react with the anti-MuLV serum (Fig. 6). Thus, these immunological tests failed to detect any production of type-C particles by these cells. Furthermore, we have not been able to detect MuLV in the tissue culture fluid by the conventional XC-test. The detection of MuMTV antgens correlates with our findings of budding B particles from the cell surface and the presence of such particles in the culture fluid. (c) Viral protein. Polyacrylamide-gel electrophoresis (Fig. 7) of the purified virus labeled with [“Hlglucosamine and ‘Clabeled amino acids showed five major polypeptides; the two major glycopeptides have molecular weights of 55,000 (~55) and 34,000 (~34). The nonglycopeptides have molecular weights of 28,000 (~281, 18,000 (~181, and 12,000 (~12). These results show that the particles produced by the MuMT73 cells are truly B particles, since identical polypeptide compositions have been described previously for type-B particles but not for C particles (Dickson and Skehel, 1974; Parks et al., 1974a; Teramoto et al., 1974; Sarkar and Dion, 1975; Kimball et al., 1976). Virus

Quanditation

In order to determine semiquantitatively how much MuMTV-specific antigen is synthesized per cell and whether the percentage of antigen-producing cells increases with time Immunofluorescence.

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FIG. 6. Immunodiffusion tests with rabbit antiMuMTV serum and rabbit anti-MuLV group-specific serum. Wells 1 and 4 contained purified virus preparation from MuMT-73 cell cultures and Rauscher leukemia virus, respectively. The central well (61 contained anti-MuMTV serum. and the peripheral well (5) contained antibody against the internal antigen of MuLV group-specific (Rauscher).

after the cells were seeded, immunofluorescence tests, both with fixed and unfixed cells, were performed. Figure 8 illustrates an example of results obtained with membrane fluorescence. It should be mentioned that there were variations in results from one experiment to the other. In general, between 0 and 3 days, approximately 25% of the cells were found to be weakly positive for MuMTV antigen(s). A sharp increase in intensity of fluorescence was observed between Days 4 and 6, and the cells maintained a high level of fluorescence intensity for a period of about 6-10 days. However, the percentage of fluorescing cells did not increase significantly during this period of maximum viral antigen synthesis; only about 35% of the cells were positive. Similar fluorescence results were obtained with acetone-fixed cells. These observations suggest that those cells (of this particular cell line) which are not good producers of MuMTV antigens remain nonproductive, while cells which were ini-

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21

MuMTV

6

4

2

Days

Oo

5

10

15

20

25

30

35

Gel Section electrophoresis of from tissue culture-grown MuMTV. Five major proteins are observed. These are designated as ~55 (55,000 MW), p34 (34,000 MW), p28 (28,000 MW). p18 (18,000 MW), and p12 (12,000 MW); p55 and p34 are glycopeptides FIG.

7. Polyacrylamide-gel

proteins

tially scored as positive by immunofluorescence increase their viral antigen production with time. It is interesting to note that mature MuMTV is being constantly produced by the producer cells and is released into the culture medium; however, the nonproducer cells, even through they are exposed to the virus, are not stimulated to produce any virus. Our findings are in agreement with the results obtained by Parks and Scolnick (1973) using another cell line. These authors have found marked variations of virus levels in cloned cells and have shown that dexamethasone can only stimulate MuMTV-RNA transcription in those cells with a basal level of MuMTV-RNA transcription (Parks et al., 1975). Particle counting. Using the negative stain drop method, we estimated the amount of virus released by the cells at various stages of cell growth after passage. The reliability of the drop method in relation to the spray method was established by comparing the results obtained by these two techniques. Figure 9 illustrates the

FIG. 8. Expression of MuMTV antigen(s) on the cell surface of MuMT-73 cells at various days after transfer. Cells from two culture flasks (75 cmr) were trypsined, pooled, and plated in equal amounts in 11 small flasks (25 cm”). For immunofluorescence tests, cells from each flask on desired days were gently scraped with a rubber policeman, washed twice with Hanks’ balanced salt solution (BSS), and dispersed by gentle pipetting in a small volume of BSS. Immunological tests with rabbit anti-MuMTV serum were performed using standard techniques. Trypsinization of cells was not done in order to avoid any presumed loss of virus-specific antigen from the cell surface. Percentage of MuMTV positive cells was estimated from a total count of 300-400 cells in each experiment.

results of particle counting of two different viral preparations at various dilutions by the spray and the drop methods, and a reasonably good correlation is seen between the amount of viral protein and the number of virus particles. Counting of virus preparations containing 10’2-10’” particles/ml or more was found to introduce an error of about 30-50% due to clumping of virus particles. Under our conditions of specimen preparation for microscopy, particle concentrations within the range of 1O”-1O11particles/ml were found to be most suitable. Uniform dispersion of virus particles (Fig. lo), which can easily be accomplished by proper dilution or concentration, is essential for the reproducibility of results in virus counting. Although particle counting by negative-staining methods has been criticized by Dubochet and Kel-

22

SARKAR.

POMENTI.

lenbarger (19721, we believe that these methods can be used with confidence, at least in the case of MuMTV, by using latex beads as indicator particles. It should be

o&k-

00 Virol

I 120 Protein (pg)

1 160

J 200

FIG. 9. Relationship between the number of MuMTV particles and the amount of protein. The average mass of protein of a particle, as estimated from the calibration curve, is 6.4 x 10 Iti g.

FIG. 10. A mixture PTA. 70,000x

of mouse

mammary

tumor

virus

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DION

mentioned that by using the drop method, Monroe and Brandt (1970) have obtained reliable results in counting murine leukemia viruses. We have estimated from Fig. 9 that an MuMTV particle contains about 6.4 x 10 I6 g of protein, a value which is in general agreement with the estimation of the mass of other viruses of the oncornavirus group (Bonar and Beard, 1959; Lyons and Moore, 1965). Figure 11 shows that the number of virus particles released into the tissue culture medium increases up to 12 days of cultivation and then declines. After an initial lag of l-3 days (or in some passages up to 5 or 6 days), the cells release virions at an increased rate. To get a reliable estimation of the number of virus particles released per cell during a 20hr period, we estimated the number of cells per flask, determined the percentage of cells positive for MuMTV antigen by and counted the immunofluorescence, number of virus particles released into the culture medium. The result of such an experiment is shown in Table 1. The amount of virus released by a mammary tumor cell per 20 hr on the seventh day in culture is nearly the same as the virus yield on the fourth day, whereas there is a 2.7-fold in-

(B) and latex

particles

tL);

negatively

stained

with

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OF TABLE

QUANTITATIVE

ESTIMATION

OF THE

MOUSE

TUMOR

Time in culture after passage (days)

4 7 10

Number

of cells (NJ

8.1 x 10” 2.5 x 10’ 2.7 x 10’

1

MAMMARY

CELLS

INTO

TUMOR

TISSUE

----__-Percentage of cells positive for MuMTV antigen” (PI 31 37 41

23

MuMTV

VIRUS

CULTURE

PARTICLES

Number of virusproducing cells IN,, = (N,, x Pi/ 1001

x 10”

RELEASED

BY MAMMARY

MEDIUM

Number of virusesh released into medium per milliliter per 20 hr (N,)

Number of viruses released” per cell per 20 hr [W,, x lOW,l 260 301 809

2.5 9.3

x 10”

6.5 x 10’ 2.8 x 10”

1.1

x 10’

8.9

x 10”

‘I Estimation by immunofluorescence test. ’ Particle counting was done by the drop method. I’ Volume of medium per flask was 10 ml.

crease in virus release on Day 10 as compared to Day 7. From Table 1 as well as from Fig. 11, it is clear that the optimum release of virus into the culture medium by l-2 x 10’ cells during a period of 20-24 hr is approximately 10y particles/ml. Similar results were obtained from a biochemical assay based on the hybridization of labeled MuMTV-specific DNA (cDNA) with the RNA extracted from virus harvested from the culture medium [accompanying paper, Sarkar et al. (197711. RDDP Amy Since RDDP activity is associated with MuMTV, we monitored RDDP activity as a sensitive measure of MuMTV expression in our cultured cells and compared these results with particle counting. Quantitation of MuMTV by RDDP assay has been used by others (Dickson, 1973; Young et al., 1975). The high speed pellet obtained from tissue culture fluid was found to contain a high level of DNA polymerase activity when assayed using the synthetic template/primer poly(rC) .oligo(dG) in the presence of Mg’+ and a low level of activity in the presence of MI?+. To determine whether or not this activity is associated with the mouse mammary tumor virus as opposed to cellular components or with type-C virus, pilot experiments were performed. Vu-us was labeled with ‘-‘C-labeled amino acids at 10 &X/ml in a tissue culture medium for 24 hr prior to harvesting. The virus-containing suspension was centrifuged on a sucrose gradient for 2 hr at

FIG. 11. Kinetics of virus release into the tissue culture medium. The number of virus particles was counted by the drop method (for description, see text), and the amount of viral reverse transcriptase (RDDP) was measured using synthetic template/ primer poly(rCi~oligo(dG1 in the presence of Mg2+ or Mn’+. The inset shows a linear relationship between the number of particles and the amount of RDDP activity. Harvesting of virus at each time point was made for a period of 24 hr from four flasks, each containing 12 ml of medium.

50.1

32,000 rpm (SW rotor), and 0.2-ml fractions were collected by bottom puncture of the gradient tube. Five-microliter aliquots from each of four consecutive fractions were pooled and assayed for DNA polymerase activity, and the remainder of each of the fractions was tested for the incorporation of “C-labeled amino acids into virus by radioactive counting. Figure 12 shows that the DNA polymerase activ-

24

SARKAR.

-RDDP

POMENTI.

/ ,

,\4 :

OOU

!5

Fraction

Number

FIG. 12. Density gradient centrifugation of I%amino acid-labeled tissue culture virus. Virus was labeled for 24 hr, harvested from one 75cm’ flask containing 10 ml of medium, and centrifuged to a pellet after removal of cell debris. The resuspended pellet was centrifuged for 2 hr in a O-60% sucrose gradient. Fractions (0.2 ml) were collected by bottom puncture of the tube; 5 ~1 of each of four consecutive fractions were pooled and assayed for reverse transcriptase activity, and the remaining portion of each of the fractions was used for estimating “C counts and for density (by refractometry) determinations. The peak of “C! counts and the RDDP activity coincided at a density of 1.17-1.18 g/cm’.

ity and ‘“C counts coincide on the gradient at a density of 1.17 g/cm”. On electron microscopic observation, the material banded at this density contained only MuMTV particles (Fig. 4). Immunocliffusion also detected only MuMTV antigen in this fraction and not MuLV (Fig. 6). The PAGE patterns of labeled proteins from these particles were observed to be of typeB particles (Fig. 7). Having established that the DNA polymerase assay detects MuMTV, the release of MuMTV into the culture fluid at various stages of cell culture was measured by the polymerase assay and compared with particle counting. Figure 11 illustrates that virus production, as measured both by particle counting and polymerase assay, increases up to 12 clays and then declines. Furthermore, Fig. 11 (inset) also demonstrates that a good correlation exists between the particle count

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and the RDDP activity. For this reason, all subsequent quantitation of virus production was based on the RDDP assay. The kinetics of virus production shown in Fig. 11 was obtained by changing the culture medium and harvesting the virus every 24 hr. Experiments were therefore designed to measure the quantity of virus accumulated in the culture medium over extended periods of time in order to optimize the yield of virus per harvest. For this purpose, an equal number of cells was seeded in each of eight flasks and fed with 20 ml of supplemented medium. The amount of virus released into the medium of each flask was monitored by RDDP assays on various days beginning 1 day after the initial transfer. Figure 13a illustrates that, if the medium was not changed, the polymerase acbivity, measured in presence of Mg”+, reached its maximum on the fifth day and then continuously declined up to the eighth day. However, the accumulation of virus in the culture medium as monitored by negative-staining electron microscopy continued during this time. It is highly probably that these disparate observations are due to the inactivation of viral polymerase at 37” in the tissue culture medium as a function of time and pH. On Day 9, the pH of the medium of the remaining culture flasks was found to be very acidic, and therefore adjustments were made under sterile conditions to bring the pH close to neutrality. As a result, an increase in viral polymerase activity was observed for a short period of time, after which the activity decreased again (Fig. 13). Membrane immunofluorescencetests revealed that the percentage of fluorescing cells and the intensity of fluorescence increased with time of culture in a manner similar to that shown in Fig. 8 and remained practically unchanged up to 13 days. Up to 9 days, the cells appeared to have normal morphology, and practically all were attached to the flask. Thereafter, some cells started to detach from the flask; at the end of 14 days about 60-70s of the cells were unattached. It should be noted that the level of RDDP activity expressed in the presence

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‘\ ‘\

?C-

:

Dc~ys af’er transfer

FIG. 13. An estimation of the amount of reverse transcriptase activity associated with the virus produced by M&IT-73 cells; the cells were maintained either in the seeding medium (a) or in medium changed several times (b). To obtain the results shown in (a), about 4 x 10’ cells were plated in each of eight 75.cm’ flasks containing 20 ml of growth medium. Virus was harvested on the days indicated and semipurified; equal amounts of virus were assayed for RDIDP activity in the presence of Mg’* or Mnr+. During the entire period of this experiment, the medium was not changed; the pH of the remaining three flasks of this series was adjusted to 7.0 on Day 9. The results in (b) (control1 were obtained by measuring the RDDP activity of semipurified virus harvested from medium changed regularly every 3 days. Two flasks were used for this set of experiments; the value shown corresponding to each time point is the average of two measurements. The enzyme activities in (a) and (bl cannot be directly compared since the number of cells and the time taken to harvest the virus varied.

of Mn’+ was consistently low compared to the level expressed in the presence of M@+, and the activity in the presence of Mn2+ increased only when the medium was depleted of nutrients. No such dispro-

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MuMTV

portionate increase in Mn”+-dependent RDDP activity was observed in the control culture (Fig. 13b). Since Mn?+ is a preferred cation for the reverse transcriptase activity of type-C particles (Howk et al ., 1973; Dion et al., 1974; Marcus et al., 1976), the increased MI?‘-dependent RDDP activity in Fig. 13a could be due to the release of type-C particles from these cells. A comparison of the results presented in Figs. 11 and 13 suggests that the quality and the amount of virus (MuMTV) that could be collected from the tissue culture medium depend upon the time of harvest. The maximum yield of virus would occur 4-6 days after the cells become confluent. If tissue culture-grown virus is to be used for RDDP activity or its products, it should not be allowed to be in medium more than 4 days. Effect

of Hormones

In order to establish whether glucocorticoid hormones stimulated the production of MuMTV in our established cell line (MuMT-73) confluent cells were treated with hydrocortisone or dexamethasone alone or together with insulin (Table 2). It was found that the addition of dexamethasone and insulin to the culture medium resulted in a 9.5-fold increase in MuMTV production, whereas dexamethasone alone increased virus production about 7.4-fold. Hydrocortisone alone or in combination with insulin also stimulated virus production (3.2- and 6.5-fold, respectively). In contrast, treatment of the cells with insulin alone did not significantly stimulate MuMTV production. Although we have not investigated the kinetics of virus induction by hormones or optimized the hormone concentration, our results, in general, conform to the observations by others that glucocorticoid hormones enhance the production of MuMTV in cultured cells (McGrath, 1971; Parks et al., 1974b, 1975; Fine et al., 1974; Dickson et al., 1974; Ringold et al., 1975; and Young et al., 1975). DISCUSSION

In order to establish a cell line that would continuously produce a large

26

SARKAR, TABLE STIMULATION HORMONES

Treatment”

OF MuMTV IN THE

POMENTI.

2 PRODUCTION

BY

MuMT-73 CELL LINE RDDP ac- Increase in tivity MuMTV (cpm)” production by hormone (n-fold)’

None Insulin (10 pgiml) Hydrocortisone (10 n.g/ml) Dexamethasone (10 pg/

6,613 11,905 21,169 48,906

3.2 7.4

42,981

6.5

62,824

9.5

1.8

ml) Insulin (10 pgiml) + hydrocortisome (10 pgiml) Insulin (10 pgiml) + dexamethasone (10 pgiml)

d Equal numbers of cells (2 x IO”) were plated from a pool of trypsinized cells (24 x 10fi) into 12 75cmL flasks containing 12 ml of basic growth medium supplemented with 15% fetal calf serum, 100 U/ml of penicillin, and 100 pg/ml of streptomycin. The medium was changed on the third and fifth days. On the seventh day, 10 of the flasks received various combinations of hormones in duplicate. Hormones in fresh medium were again added to these flasks on the ninth day. Culture fluids were collected after 24 hr. Each flask was washed twice, the cells were trypsinized, and the total number of cells in each flask was estimated by cell counts. ’ Semipurified virus was assayed in the presence of Mg+’ for reverse transcriptase (RDDP) activity (see Materials and Methods). The counts shown were normalized for 9 x lOti ceils, since there were variations in the total number of cells in different flasks. Each count represents the average of two measurements from duplicate flasks. ’ Ratio of counts per minute (RDDP activity) obtained with treated cultures to counts in the control.

amount of mouse mammary tumor virus (MuMTV or B particles), we used spontaneous mammary tumors from a variety of mouse strains. After seeding the trypsinized tumor cells in culture, the amount of B particles produced by these cells was monitored by negative stain electron microscopy. In general, in all the various murine mammary tumor cells cultured, numerous B particles (5-10 particles/grid square) were usually observed in a 50- to loo-fold concentrate of the supernate collected from primary cultures. After subculturing, the B particle production varied widely and in a most unpredictable way from one culture to the next. By two or

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three passages, most of our cultures derived from GR, RIII, and A tumors started producing low amounts of type-C virus particles with a gradual decrease in type-B particle production, and therefore all these cultures were discarded. The only cell line that we have been able to establish and that consistently produces a large amount of only type-B virus particles was from BALB/cfC3H mice. This does not necessarily indicate that the SUCcess of establishing a cell line with high virus production depends upon the strain of mice, since virus-producing cell lines from GR and RI11 as well as C3H strain mice have been established by others (Lasfargues et al., 1972; Owens and Hackett, 1972; Ringold et al., 1975). It is rather a general phenomenon that many tumors vary with respect to type-B as well as typeC particle expression, and the preference of the type-B and/or type-C particle production varies greatly and unpredictably from one culture to the other (Sanford et al., 1961). It should be mentioned, however, that although the type-C virus particle genome is ubiquitous in mice (Huebner and Todaro, 1969; Todaro and Huebner, 1972), the proliferation of B particles in mammary tumor cultures is predominant soon after culturing, while C particle production is a late event (Dmochowski et al., 1971; Yagi, 1975; Owens and Hackett, 1972), and in some cultures it is practically nonexistent (Parks and Scolnick, 1973; Owens and Hackett, 1972; Lasfargues et al., 1972; Ringold et al., 1975). These observations suggest that the mammary epithelium is preferred for B particle production as compared to type-C particles. However, type-B virus or viral antigens have been seen occasionally in other organs of mice in addition to mammary tissues [for details, see Nandi and McGrath (1973) and two recent reports by Gillette et al. (1974) and Rongey et al. (197511. That our established cell line produces only type-B particles under normal conditions of cell culture is based on: (i) The buoyant density of the particles as measured in a sucrose gradient is 1.17 g/cm:’ (Fig. 12). In a cesium chloride gradient, these particles band at a density of 1.21 g/

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cti’ (data not shown), a characteristic property of type-B particles but not of typeC particles, which have a similar buoyant density in both salt solutions (Sarkar and Moore, 1974). (ii) The morphology of the budding particles as well as of the extracellular particles is consistent with that of type-B particles. The surface of the budding virions contains projections; extracellular mature virions always have an eccentric nucleoid surrounded by an envelope from which projections protrude (Bernhard, 1960). (iii) Cells treated with rabbit anti-MuMTV (prepared against ether-disrupted virus) serum produced positive immunofluorescence, whereas antiMuLV serum failed to detect any cellassociated MuLV antigens. The crude viral preparation from the culture fluid and the sucrose density gradient-purified viral preparations produced immunoprecipitation lines in a micro-Ouchterlony test with anti-MuMTV serum but not with anti-MuLV serum. (iv) SDS-PAGE analysis of the glucosamine and amino acidslabeled virus revealed that the proteins and glycos,ylated proteins of the virions were simil,ar to those of the milk-borne viruses (Parks et al., 1974a; Dickson et al., 1974; Sarkar et al., 1977) as well as of virus harvested from short-term cultivation of mammary tumor cells from a variety of mouse strains (Teramoto et al., 1974; Dickson and Skehel, 1974; Sarkar and Dion, 1975; Kimball et al., 1976). As previously reported, the polypeptides composing the type-B virus particles are substantia.lly different from the protein components of type-C viruses (Nowinski et al., 1972). (v) High speed pellets of the culture fluid and semipurified and/or purified virus contained RNA-instructed DNA polymerasle activity with a marked preference for MgZ+, a characteristic property of type-B particles (Howk et al., 1973; Dion et al., 1974; Marcus et al., 1976). (vi) Finally, our examination of the culture fluid for the presence of murine leukemia virus by the XC test was negative. It should be mentioned, however, that of all the tests used in this study for detecting MuLV, immunodiffusion is the least sensitive and that the XC test would not exclude the possibil-

OF

MuMTV

27

ity of xenotropic (Levy, 1973) or endogenous ecotropic (Hopkins and Jelicoeur, 1975; Rapp and Nowinski, 1976) type-C viruses being expressed in our cell line. Although the extracellular virions produced by the cultured cells were typical of type-B particles, some differences were observed between the morphogenesis of these particles and of those usually found in spontaneous primary mammary tumours. In the latter case, doughnutshaped type-A particles assemble within the cytoplasmic matrix, and after assembly they presumably move to the periphery of the cell and make intimate contact with the plasmalemma or with the membrane surrounding the cytoplasmic vacuoles prior to budding (Bernhard, 1960). Similar viral morphogenesis can be assumed in a number of cell lines described previously since replication of A particles was observed in those cells (Lasfargues et al., 1974, Owens and Hackett, 1972; Dmochowski et al., 1971; Hoshino and Dmochowski, 1973; Yagi et al., 1973). The cell line described here continuously produced abundant B particles, but no type-A particles were found. These observations suggest that the accumulation of type-A particles within the cytoplasm is not a necessary factor for the morphogenesis of B particles in cultured cells. Our results show that the assembly of A particles can occur near the cell membrane. It might be the rule rather than the exception that only those A particles which are assembled near the cell membrane (observed in this study as well as in tumors) can successfully bud off to form B particles and that the accumulated intracytoplasmic A particles (seen invast excess in tumors) do not migrate to the cell membrane to be incorporated into the development of B particles. Although our observations would support such a hypothesis, the actual mechanism of MuMTV morphogenesis remains unknown. Another important aspect of type-B particle morphogenesis which we have observed in these cells is that a significant proportion of the particles initiates budding in a manner similar to the budding of type-C particles; i.e., the nucleocapsid starts forming as a double-

28

SARKAR,

POMENTI,

shelled structure (crescent-like structure) below the site of viral budding. But unlike the classical type-C particles (de Harven, 1974), the membranes of these buds were found to acquire spikes. This form of budding is not the usual mode of MuMTV morphogenesis, although it has been observed occasionally in primary tumors (Dalton and Potter, 1968; Sarkar and Moore, 1972). It was suggested earlier (McGrath, 1971; McGrath et al., 1972) that the organization of mammary tumor cells into domes is required for MuMTV replication. By the immunofluorescence test, we observed that MuMTV antigens were present in the dome cells as well as in the monolayer cells. The intensity of fluorescence was greater in the dome cells than in the nondome cells, but this difference is most likely due to the difference in cell density. These results suggest that the production of MuMTV, at least in the case of our established cell line, is not restricted to the dome cells alone. Furthermore, we found that doming is dependent upon the seeding density of the cells. Parks and Scolnick (1973) have compared the amount of MuMTV expression by two mammary tumor cell lines which they cloned from Sykes’ CCL-51 line (Sykes et al., 1968). Although the morphological characteristics, including doming, of these two lines were similar, the lines differ markedly in their expression of virus information. From these results, Sykes et al. concluded that the ability of mammary tumor cells to form domes is not sufficient for the production of MuMTV antigens or particles. Recently, Das et aZ. (1974) have shown that doming is not a special characteristic of neoplastic mammary epithelium alone; normal epithelium can produce domes equally well. Thus our results and those of Parks and Scolnick (1973) and Das et al. (1974) suggest strongly that (i) dome formation by mouse mammary epithelium is not a prerequisite for MuMTV production; and (ii) doming is dependent upon the seeding density of epithelial cells (although this may not be the only factor), normal or neoplastic, maintained in primary cultures as well as in established cell

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lines. However, the mechanism of cellular recognition in dome formation remains unknown. ACKNOWLEDGMENTS Thanks are due G. S. Fout and E. Whittington for technical assistance and Dr. A. J. McClelland for editorial help. This research was supported in part by NC1 Grants CA-17129, CA-08740, CA-08748, and CA-11405. REFERENCES BERNHARD, W. (1960). The detection and study of tumor viruses with the electron microscope. Cancer Res. 20, 112-727. BONAR, R. A., and BEARD, J. W. (1959). Virus of avian myeloblastosis. XII. Chemical constitution. J. Nat. Cancer Inst. 23, 183-197. CARDIFF, R. D.. BLAIR, P. B., and DE OME, K. B. (19681. In vitro cultivation of the mouse mammary tumor virus: Replication of MTV in tissue culture. Virology 36, 313-317. DALTON, A. J., and PORTER, M. (19681. Electron microscopic study of the mammary tumor agent in plasma cell tumors. J. Nat. Cancer Inst. 40, 13751385. DAS, N. K., HOSIK, H. L., and NANDI, S. (19741. Influence of seeding density on multicellular organization and nuclear events in cultures of normal and neoplastic mouse mammary epithelium. J. Nat. Cancer Inst. 52, 849-855. DE HARVEN, E. (1974). Remarks on the ultrastructure of type A, B, and C virus particles. In “Advances in Virus Research” (M. A. Lauffer et al., eds.), Vol. 19. pp, 221-264. Academic Press, New York. DICKSON, C. (1973). Mouse mammary tumor virus RNA-dependent DNA polymerase: Requirements and products. J. Gen. Virol. 20, 243-247. DICKSON, C., HASLAM, S., and NANDI, S. (1974). Conditions for optimal MTV synthesis in vitro and the effect of steroid hormones on virus production. Virology 62, 242-252. DICKSON, C.. and SKEHEL, J. J. (1974). The polypeptide composition of mouse mammary tumor virus. Virology 58, 387-395. DION, A. S., VAIDYA, A. B., and FOUT, G. S. (1974). Cation preferences for poly(rC) oligo(dG)-directed DNA synthesis by RNA tumor viruses and human milk particles. Cancer Res. 34, 3509-3515. DMOCHOWSKI, L., WILLIAMS, W. C., SWEARINGEN, G. R., MYERS, B., and FUJINAGA, S. (1971). Immunological studies on mouse mammary tumors and leukemia. Tex. Rep. Biol. Med. 29, 41-62. DUBOCHET, J., and KELLINBARGER, E. (1972). Selective adsorption of particles to the supporting film and its consequence on particle counts in electron microscopy. Microsc. Acta 72, 119-130.

REPLICATION FINE, D. L., PI.OWMAN, J. K., KELLEY, S. 9., ARTHUR, L. O., and HILLMAN, E. A. (19741. Enhanced production of mouse mammary tumor virus in dexamthasone-treated, 5-iododeoxyuridinestimulated mammary tumor cell cultures. J. Nat. Cancer Inst. !i2, 1881-1886. GILLETTE, R. PI., ROBERTSON, S., BROWN, R., and BLACKMAN, K. E. (1974). Expression of mammary tumor virus antigen on the membrane of lymphoid cells. J. PJat. Cancer Inst. -53, 499-505. HARTLEY, J. W , ROWE, W. P., and HUEBNER, R. J. (1970). Host-range restrictions of murine leukemia viruses in mouse embryo cell cultures. J. Vi&. 5, 221.-22. HILGERS, J., NOWINSKI, R. C., GEERING, G., and HARDY, W. (1972). Detection of avian and mammalian oncogeneic RNA viruses (oncornaviruses) by immunofluorescence. Center Res. 32,98-106. HOPKINS, N., and JOLICOEUR, P. (19751. Variants of N-tropic leukemia virus derived from BALBic mice. J. Viral. 16, 991-999. HOSHINO, M., and DMOCHOWSKI, L. (19731. Electron microscope study of antigens in cells of mouse mammary tumor cell lines by peroxidase-labeled antibodies in sera of mammary tumor-bearing mice and of patients with breast cancer. Cancer Res. 33, 2551.-2561. HOWK, R. S., RYE, L. A., KILLEEN, L. A., SCOLNICK, E. M., and PARKS, W. P. (1973). Characterization and separation of viral DNA polymerase activities in mouse milk. Proc. Nut. Acad. Sci. USA 70, 2117-2121. HUEBNER, R.
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neous ecotropic mouse type C viruses deficient in replication and production of XC plaques. J. Vird. 17, 411-417. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Bid. 17, 208-212. RINGOLD, G., LASFARGUES, E. Y., BISHOP, J. M., and VARMUS, H. E. (1975). Production of mouse mammary tumor virus by cultured cells in the absence and presence of hormones: Assay by molecular hybridization. Virology 65, 135-147. RONGEY, R. W., ABTIN, A. H., ESTES, J. D., and GARDNER, M. B. (1975). Mammary tumor virus particles in the submaxillary gland, seminal vesicle, and nonmammary tumors of wild mice. J. Nat. Cancer Inst. 54, 1149-1156. ROWE, W. P., HARTLEY, J. W., and PUGY, W. E. (19’70). Plaque assay technique for murine leukemia viruses. Virology 42, 1136-1139. SANFORD, K. K., ANDERVONT, H. B., HOBBS, E. L., and EARLE, W. R. (1961). Maintenance of the mammary-tumor agent in long term cultures of mouse mammary carcinoma. J. Nat. Cancer Inst. 26, 1185-1191. SARKAR, N. H., and DION, A. S. (1975). Polypeptides of the mouse mammary tumor virus. I. Characterization of two group-specific antigens. Virology 64, 471-491. SARKAR, N. H., and MOORE, D. H. (1972). Electron microscopy in mammary cancer research. J. Nat. Cancer Inst. 48, 1051-1058. SARKAR, N. H., and MOORE, D. H. (1974). Separation of B and C type virions by centrifugation in gentle density gradients. J. Virol. 13, 1143-1147. SARKAR, N. H., POMENTI, A. D., and DION, A. S. (1977). Replication of mouse mammary tumor vi-

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rus in tissue culture. II. Kinetics of virus production and the effect of RNA and protein inhibitors on viral synthesis. Virology 77, 31-44. SHAPIRO, D.. BRANDT, W. E.. CARDIFF, R. D., and RUSSELL, P. K. 11971). The protein of Japanese encephalitis virus. Virology 44, 108-124. SHIGEMATSU, T., DMOCHOWSKI, L., and WILLIAMS, W. C. (1971). Studies on mouse mammary tumor virus I MTV) and mouse leukemia virus (MuLV) by immunoelectron microscopy. Cancer Res. 31, 20852097. SYKES, J. A., WHITESCARVER, J., and BRIGGS, L. (1968). Observations on a cell line producing mammary tumor virus. J. Nat. Cancer Inst. 41, 1315-1327. TERAMOTO, Y. A., PUENTES, M. J., YOUNG, L. J., and CARDIFF, R. D. (1974). Structure of the mouse mammary tumor virus: Polypeptides and glycoproteins. J. Viral. 13, 411-418. TODARO, G. J., and HUEBNER, R. J. (1972). The viral oncogene hypothesis: New evidence. Proc. Nat. Acad. Sci. USA 69, 1009-1015. WATSON, D. H., RUSSELL, W. C., and WILDY, P. (1963). Electron microscopic particle counts on herpes virus using the phosphotungstate negative staining technique. Virology 19, 250-260. YAGI, M. J. (19’73). Cultivation and characterization of BALBlcfC3H mammary tumor cell lines. J. Nat. Cancer Inst. 51, 1849-1860. YAGI, M. J. (1974). Further observations on the production of oncornaviruses in MJY-alpha cell line. J. Nat. Cancer Inst. 53, 1383-1385. YOUNG, L. J., CARDIFF, R. D., and ASHLEY, R. L. (1975). Long-term primary culture of mouse mammary tumor cells: Production of virus. J. Nat. Cancer Inst. 54, 1215-1220.