In Vitro System for Production of Mouse Mammary Tumor Virus

An in vitro system for production, purification, and concentration of mouse mammary tumor virus is described. Monolayer cultures of C3H mouse mammary tumor cells propagated at 34 C in roller bottles in the presence of dexamethasone, a glucocorticoid hormone, release B-type particles which possess ribonucleic acid and a ribonucleic acid-dependent deoxyribonucleic acid polymerase. One thousandfold concentration by ultracentrifugation with subsequent gradient fractionation yielded > 7 × 1010 particles per ml in the 1.16- to 1.18-g/ml region. Mouse mammary tumor virus produced in this system was free of detectable C-type virus.

An in vitro system for production, purification, and concentration of mouse mammary tumor virus is described. Monolayer cultures of CH mouse mammary tumor cells propagated at 34 C in roller bottles in the presence of dexamethasone, a glucocorticoid hormone, release B-type particles which possess ribonucleic acid and a ribonucleic acid-dependent deoxyribonucleic acid polymerase. One thousandfold concentration by ultracentrifugation with subsequent gradient fractionation yielded > 7 x 1010 particles per ml in the 1.16to 1.18-g/ml region. Mouse mammary tumor virus produced in this system was free of detectable C-type virus.
As a virus reagent, mouse mammary tumor virus (MMTV) is obtained from milk, mammary tumors, or from explants or continuous cell lines derived from mammary tumors of inbred strains of mice. Although mouse milk and tumors yield large numbers of virus particles, these materials are generally contaminated with lipid or protein, whereas MMTV produced from tissue culture is relatively free of impurities. Over the past 21 years, reports from a number of laboratories have described systems for in vitro production of MMTV (2, 5-8, 17, 18, 21). In general, each has been unsuccessful, due to contaminating type-C virus (5,7), finite life span of the cultures (2,6,17) or decreased virus production (8,18,21). Utilizing the continuous cell line derived from a C,H mouse mammary tumor, described by Owens and Hackett (14), we have developed an in vitro system for intermediate or large-scale production of MMTV. In this system, MMTV production is enhanced by dexamethasone (DXM), a synthetic glucocorticoid, as demonstrated previously by our laboratory (3) and that of Parks et al. (15). MATERIALS (5 ACi/ml). Culture fluids were processed as above, and the virus pellets were resuspended in TNE buffer then layered over 20 to 55% continuous sucrose gradients. The virus was sedimented to buoyant density equilibrium by centrifugation at 80,000 x g in a Beckman SW41 rotor. The gradients were fractionated by puncture of the tube bottom and the radioactivity was determined by assaying 0.4 ml of each fraction. Scale-up and purification of MMTV. Scale-up of Mm5mt/c, cells from seed ampoules is shown in Fig. 1. For production, the Mm5mt/c1 cells were propagated in roller bottles (Bellco, Vineland, N.J.). Small (10 by 25 cm) and large (10 by 50 cm) bottles were seeded with 1.5 x 107 cells and 3.0 x 107 cells, respectively. Bottles were incubated at 34 C on roller racks (0.5 rpm) and medium was changed every 3 days (125 ml/small roller bottle and 250 ml/large roller bottle). Spent medium was stored at -70 C until used. The medium was thawed, clarified at 8000 x g (100 ml/min) in a continuous-flow Sorvall SS-4 centrifuge with the Szent-Gyorgi and Blum system, and banded on a 20 to 60% continuous sucrose gradient with an RK ultracentrifuge using an RK-3 rotor (Electronucleonics, Inc., Rockville, Md.) at 90,000 x g. Equivalent results were obtained when clarification and concentration were performed concurrently using an RK-6 rotor at 90,000 x g. The gradient was unloaded, monitored at 260 and 280 nm, and the refractive index of each 50 ml fraction was determined. Those fractions which absorbed at 260 and 280 nm and had a density of 1.15 to 1.19 g/ml were pooled, diluted to 28% sucrose with TNE, and pelleted at 100,000 x g for 1 h in a Beckman L2-75B centrifuge using a type 35 rotor. The pellets were resuspended at either x100 or x1,000 in TNE and frozen at -70 C.  treated and IUdR/DXM-treated cultures was replaced with fresh DXM-treated medium. Control cultures were untreated except for medium change concurrent with the treated cultures. Supernatant fluids (approximately 100 ml) were collected on days 3 and 5 after IUdR treatment and assayed for numbers of virus particles and presence of RDDP as described in Materials and Methods. The results of those assays are presented in Table 1. On the basis of these results, it was concluded that IUdR is not required for production of MMTV in DXMtreated cultures. Furthermore, no enhancement was obtained with IUdR over that of DXM alone. These findings agree with those of Parks et al. (15).

RESULTS
Stability of DXM in growth medium. Medium containing DXM was stored at 4 C and checked for virus stimulatory activity over a 2-month period. Medium with freshly added DXM served as a control (Fig. 2). No significant differences in membrane immunofluorescence, particulate RDDP activity was noted with fresh DXM as compared to aged DXM. Similarly, while no difference was observed in virus particle count in fresh or 6-week-aged DXM, a slightly higher particle count (0.7 log10) was observed in fresh as compared to 9-week-aged DXM. Although this difference might be attributed to breakdown of DXM due to storage, it is just as likely accounted for by experimental error in sampling and electron microscope preparation and counting. These data indicate that, in an MMTV production scheme, growth medium containing DXM could be prepared in advance and stored at least 9 weeks before use.
Effect of cell seeding in the presence of  DXM. Cells were seeded in the presence and absence of DXM and allowed to attach for 24 h at 37 C. The spent medium was removed and replaced with medium containing DXM. Replicate flasks were assayed at 2-day intervals post-seeding. As demonstrated in Fig. 3, the presence of DXM at the time of cell seeding had no effect on either cell growth, particulate RDDP activity, or virus particle production in culture fluids. Higher RDDP activities and virus particle counts were observed on days 5 and 7 in cultures seeded in the presence of DXM than in those seeded in the absence of DXM, suggesting an earlier response of MMTV production in the former cultures. These results indicated that, in a production scheme, the DXM could be included in the growth medium at the time of cell seeding, thus eliminating a procedural step of adding DXM to the cultures after cell attachment. Scale-up of MMTV in roller bottles. Mm5mt/c1 cells from 75-cm2 flasks were seeded in small roller bottles and incubated in the presence of DXM until confluent. During this time cell numbers increased from 1.5 x 107 to 1.8 x 108 per bottle. Supernatant fluids were harvested and processed for virus particles and particulate RDDP (data not shown). Cells were trypsinized and a portion was assayed for cellassociated MMTV-specific antigens by membrane immunofluorescence. The remaining cells were then transferred, in scale-up, to 20 large roller bottles and incubated in medium containing DXM. Medium was changed at 3-day intervals and pooled before concentration and purification as described in Materials and Methods. The results of this production run are presented in Table 2. A major peak with a 260-nm absorbance was found in the sucrose gradient with a density of 1.156 to 1.189 g/ml (Fig. 4). This region, when fractionated, contained appreciable particulate RDDP activity (Table 2). Significantly high incorporation was observed with the dG .rC template, considered more specific for virusrelated RDDP (20), whereas low incorporation was observed with the oligo dT template, indicating low terminal transferase activity (11). The x 1000 concentrated material contained 0.7 mg of protein per ml.
A duplicate sample of this material was found to contain 7.6 x 1010 virus particles per ml. Comparable particle counts were obtained in another production run of 6.5 liters ( Table 2). Examination of negative-stained preparations (Fig. 5) revealed pleomorphic-tailed particles, having surface spikes, measuring 105 nm in average diameter, and typical of the B-type particles described in mouse milk (10,13,19).
Density of MMTV produced in vitro. Material collected from supernatant fluids from 7-day DXM-treated Mm5mt/c, cells incubated in the presence of [3H ]uridine was found to contain a peak of radioactivity at the 1.16-g/ml region of a sucrose gradient (Fig. 6). Material contained in this fraction was sensitive to digestion with ribonuclease, indicating the presence of ribonucleic acid. No significant radioactivity was observed in the 1.16-g/ml region of the gradient in supernatant material from nontreated Mm5mt/c1 cells.
Immunological reactions of MMTV produced in vitro. To determine whether MMTV produced from Mm5mt/c, cells contained antigens common to MMTV from mouse milk or the major group-specific antigen of murine leukemia virus, immunodiffusion tests were performed. Antisera to MMTV and a 27,000molecular-weight peptide (p27) isolated from MMTV virus obtained from R111 mouse milk (supplied by Wade Parks) and antiserum against murine (Rauscher) leukemia virus (RLV) p30 (gs,) (supplied by D. Fish) were tested against lysed MMTV produced from Mm5mt/c, cells. A single precipitin line (Fig. 7) was observed between the antiserum to MMTV p27 and the MMTV from Mm5mt/c cells. Whereas a precipitin line formed between RLV p30 and the corresponding antiserum to RLV p30, no line of precipitation was observed between the tissue culture-produced MMTV and the antiserum to RLV p30.
Virus samples were sent to D. Bolognesi for estimation, by radioimmunoassays, of possible murine leukemia virus proteins (p30 and gp 71). Bolognesi reported the results of the assay for the presence of gp 71 as negative and only a slight blocking (2 ng/10 ,liters of sample) was noted for the presence of the p30 protein.
Discrepancies between the p30 and gp 71 radioimmunoassays may be based on the degree of specificity of the respective proteins.   described here has one major advantage over that of milk-borne virus, namely absence of detectable type-C virus particles. The problem of contaminating type-C virus was noted in a report by Howk et al. (4) where milk from R,1, mice contained two distinct RDDP's, one of which corresponded immunologically as well as biochemically to the enzyme from type-C virus. Similarly, in a recent study in which MMTV antigens were purified from RI,, mouse milk, Parks et al. (16) state, "The goat antiserum prepared against MMTV p27 showed significant cross-reaction with gp 52 (SI) and a low level reaction with MuLV p30. This was most likely due to contamination of MMTV preparations with murine type-C virus." These studies show the need for MMTV, free of type-C virus, for viral-component antisera preparation. In the initial characterization of the Mm5mt/c1 culture, immunological as well as electron microscope examinations failed to reveal any type-C virus in the cells (14). Data presented here, in addition to electron microscope examination, fail to demonstrate the presence of type-C virus in either the Mm5mt/c, cells or virus product.
Qualitatively, Mm5mt/c ,-produced MMTV compares morphologically, immunologically, and biochemically with type-B particle preparations from MMTV bearing mouse milk. Studies are in progress to determine whether the virus produced from Mm5mt/cl cells is infectious for either mice or murine cell cultures. In preliminary experiments in which weanling female BALB/c mice were inoculated with serial dilutions (10' to 10-') of Mm5mt/ c l-derived virus, 5 of 10 mice demonstrated mammary tumors within 9 months postinoculation. The average incidence of spontaneous tumors in the BALB/c colony is 3% over a period of 15 months.