Production of the mouse mammary tumor virus in cultures of BALB/cfC3H strain

The mouse mammary tumor virus (MTV) reproduces by a budding mechanism at the cell membrane of mouse mammary epithelial cells. In tissue culture, the tumor cells release their virions in the culture supernatant from which they can be removed by high speed centrifugation. Mammary tumor cells from the RIII, GR, and A strains of mice generally produce yields of virus which decrease after a few months. Cells derived from a spontaneous mammary tumor in a BALB/cfC3H mouse have shown the capability to shed relatively large amounts of virus continuously. A quantitative estimation by membrane immunofluorescence of the number of virus producing cells in one-year-old cultures revealed the presence of viral antigen on 80 to 90% of the cells; by comparison, cultures from other mouse strains had a ratio of only 10 to 15% virus producing cells. High speed centrifugation pellets obtained from 50 ml culture supernatant provided large amounts of mature virus particles which have been characterized by electron microscopy.     

BALB/Mtv-null mice responding to strong mouse mammary tumor virus superantigens restrict mammary tumorigenesis

The absence of endogenous mouse mammary tumor viruses (MMTVs) in the congenic mouse strain, BALB/Mtv-null, restricts the early steps of exogenous C3H MMTV infection, preventing the superantigen (Sag) response and mammary tumorigenesis. Here we demonstrate that BALB/Mtv-null mice also resist tumor induction by FM MMTV, which encodes a stronger Sag compared to C3H MMTV. In contrast to infections with C3H MMTV, Mtv-null mice show FM-MMTV Sag-specific responses comparable to those observed in susceptible BALB/c mice. Neither virus shows significant replication in the spleen or mammary gland. Thus, Mtv-null mice restrict MMTV replication and mammary tumorigenesis even after a robust Sag response.     

Identification of a precursor protein to the major glycoproteins of mouse mammary tumor virus.

Mouse mammary tumor virus-producing cultures of mouse mammary tumor cells synthesize a viral-related polypeptide of molecular weight of 73,000 (gp 73) which is rapidly labeled during a short pulse but disappears during the chase concomitantly with the appearance of label in the virion glycoproteins gp 49 and gp 37.5/33.5. The addition of the protein synthesis-inhibitor cycloheximide to the chase medium has little effect on this conversion. Treatment of the proposed precursor with alpha-chymotrypsin leads to the formation of a polypeptide of molecular weight 49,000, similar to the major virion glycoprotein. A comparison of tryptic digest maps of the glycoproteins involved supports the hypothesis that both the viral glycoproteins gp 49 and gp 37.5/33.5 are derived from gp 73.     

Two genetically transmitted BALB/c mouse mammary tumor virus genomes located on chromosomes 12 and 16.

We have examined EcoRI-restricted cellular DNA from BALB/c mouse-hamster somatic cell hybrids by blot hybridization for the presence of mouse mammary tumor virus-related sequences. Results of this analysis show that mouse mammary tumor virus-related proviral copies are located on chromosomes 16 (16-kilobase-pair fragment) and 12 (10.5- and 7.7-kilobase-pair fragments).     

Organization of mouse mammary tumor virus-specific DNA endogenous to BALB/c mice.

We used restriction endonucleases to prepare physical maps of the mouse mammary tumor virus (MMTV)-specific DNA endogenous to the BALB/c mouse strain. The mapping was facilitated by the DNA transfer procedure, using complementary DNAs specific for the whole and for the 3' terminus of MMTV RNA to detect fragments containing viral sequences. The strategies used for the arrangement of fragments into physical maps included sequential digestions with two or three enzymes; preparative isolation of EcoRI fragments containing viral sequences; and comparisons of virus-specific fragments derived from the DNA of several mouse strains. Most of the MMTV-related DNA in the BALB/c genome is organized into two units (II and III) which strongly resemble proviruses acquired upon horizontal infection with milk-borne strains of MMTV and other retroviruses. These units contain approximately 6.0 x 10(6) Mr of apparently uninterrupted viral sequences, they bear redundant sequences totaling at least 700 to 800 base pairs at their termini, and the terminal redundancies include sequences derived from the 3' end of MMTV RNA. Units II and III are closely related in that they share 12 of 14 recognition sites for endonucleases, but cellular sequences flanking units II and III are dissimilar by this criterion. The remainder of the MMTV-related DNA endogenous to BALB/c mice is found in a single subgenomic unit (unit I) with a complexity of ca. 2 x 10(6) Mr; the structure of this unit has not been further defined. These results support the hypotheses that endogenous proviruses have been acquired by infection of germinal tissues with MMTV. The physical maps are also useful for identifying the MMTV genomes endogenous to BALB/c mice in studies of the natural history of mammary tumorigenesis.     

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium.

Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C3H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.     

Hormonal regulation of murine mammary tumor virus RNA expression during mammary tumorigenesis in BALB/c mice.

The effects of glucocorticoids and prolactin on murine mammary tumor virus (MuMTV) RNA expression in preneoplastic outgrowth lines and mammary tumors in BALB/c mice were investigated. Hyperplastic alveolar nodules (HAN) and a ductal hyperplasia (DH) are induced in virgin BALB/c mice by prolonged hormonal stimulation or treatment with 7,12-dimethylbenz(a)anthracene or both. Mice bearing HAN or DH outgrowth lines and mammary tumors that arose from the outgrowth lines were treated with glucocorticoids or prolactin. MuMTV RNA was quantitated by hybridization with a representative complementary DNA probe specific for MuMTV RNA. Prolactin treatment did not increase MuMTV RNA in the BALB/c HAN or DH outgrowth lines or tumors. MuMTV RNA increased after glucocorticoid treatment in the C3, C4, and C5 HAN outgrowth lines and in tumors that arose from the D1, D2, C4, and C5 HAN and CD8 DH outgrowth lines. No increase in MuMTV RNA with glucocorticoid treatment was observed in the D1 or D2 HAN outgrowth line, in the CD8 DH outgrowth lines, and in tumors that arose from the C3 HAN outgrowth line. The ability of glucocorticoids to stimulate MuMTV expression was specific since the response was dose dependent and specific for glucocorticoid hormones. Glucocorticoid treatment did not increase the level of type C viral RNA in the majority of hormone- or 7,12-dimethylbenz(a)anthracene-induced HAN outgrowth lines or tumors. These observations suggested that glucocorticoids may influence MuMTV expression during mammary tumorigenesis in BALB/c mice.     

Induction of mouse mammary tumor virus RNA in mammary tumors of BALB/c mice treated with urethane, X-irradiation, and hormones.

The involvement of mouse mammary tumor virus (MTV) in the development of mammary tumors of nonviral etiology in BALB/c mice was studied by measuring the levels of MTV RNA, MTV DNA, and MTV proteins in spontaneously arising and hormonally, chemically, and/or physically induced mammary tumors of BALB/c females. The following results were obtained. (i) Spontaneous mammary tumors contained very low levels of MTV RNA; 4 X 10(-6)% of the the cytoplasmic RNA was MTV RNA. No MTV proteins could be demonstrated by using sensitive radioimmunoassays for MTV proteins p27 and gp52. (ii) Mammary tumors induced by treatments with urethane or X-irradiation alone contained higher levels of MTV RNA; these tumors contained 3- and 19-fold more MTV RNA, respectively, compared with spontaneous mammary tumors. (iii) Mammary tumors induced by combined treatment with urethane and X-irradiation expressed high levels of MTV RNA in the mammary tumors; a 1,724-fold increase in MTV RNA content compared with spontaneous mammary tumors was observed. However, very low levels of MTV proteins gp52 and p27 were detected, suggesting some kind of impairment at the translation of the MTV RNA. MTV RNA was also induced by this treatment in mammary glands and spleens, but not in the livers of tumor-bearing animals. (iv) Balb/c females continuously exposed to prolactin contained high levels of MTV RNA and MTV proteins in stimulated mammary glands and in the hormonally induced mammary tumors. These findings suggest that MTV is not responsible for the maintenance and probably also not for the development of all murine mammary cancers.     

Identification of mouse mammary tumor virus-specific mRNA.

Complementary DNA corresponding to the RNA genome of mouse mammary tumor virus was used to identify viral RNA contained in polysomes of a virus-producing mammary tumor cell line. Separation of polysomal mRNA by agarose gel electrophoresis, transfer of the RNA to diazobenzyloxymethyl paper, and hybridization with 32P-labeled mouse mammary tumor virus complementary DNA revealed three viral RNA size classes of 10, 8.8, and 4.4 kilobases in length, respectively.     

Direct detection of exogenous mouse mammary tumor virus sequences in lymphoid cells of BALB/cfC3H female mice.

The presence of exogenous mouse mammary tumor virus (MMTV) (C3H) DNA sequences in lymphoid tissue (spleen, bone marrow, and thymus) and nonlymphoid tissue (liver and kidney) of BALB/cfC3H female mice was directly assessed by DNA hybridization methods. Lymphoid tissues were found positive for integrated MMTV(C3H) sequences in females as young as 4 weeks. In most samples, the level of splenic MMTV(C3H) infection was low (2 to 5%). Infection remained throughout the life of the animal. The percentage of spleen samples found positive for exogenous viral infection was significantly higher in females bearing mammary tumors, whether virgin or multiparous. Liver and kidney DNAs were negative for exogenous MMTV sequences, suggesting tissue type selectivity in MMTV infection.     

Development of a mouse mammary tumor virus-negative mouse strain: a new system for the study of mammary carcinogenesis.

All inbred strains of mice transmit one or more copies of mouse mammary tumor virus (MMTV) DNA integrated as proviral sequences. This complicates efforts to define viral-induced mammary carcinogenesis. Here we report the use of surgical nonlethal splenectomy in tissue typing mice and the development of an MMTV-negative mouse strain. The MMTV-negative strain allows study of the involvement of non-MMTV genes in mammary carcinogenesis. In addition, it can be used as a sterile background into which MMTV variants can be introduced. Through the techniques described here, mice containing single MMTV loci or specific combinations can be specially chosen and rapidly developed. In this manner, the oncogenecity of particular MMTV variants may be assessed.     

Isolation of a pathogenic clone of mouse mammary tumor virus.

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.     

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium

Summary Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C₃H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.     

Identification of the mammary tumor virus envelope glycoprotein (gp52) on mouse mammary epithelial cell surface.

Lactoperoxidase radioiodination of mammary epithelial cells cultured in monolayers followed by SDS-PAGE analysis revealed only a few distinct peaks. One of these, identified as major envelope glycoptrotein (gp 52) of MTV, is present on the surface of mammary epithelial cells (both tumor and normal) from chronically infected BALB/cfC3H mice but not on the surface of normal mammary epithelial cells from virus-free $\text{sol}\text{BALB}\text{c}$ mice. Its presence on the cell surface is influenced by both hormones and cell density, the same factors which greatly control the production and release of intact MTV virions into culture media. This suggests a correlation between abundance of radioiodinatable gp 52 on the cell surface and MTV found in culture media.     

Effect of trypsin on mouse mammary tumor virus.

Undisrupted mouse mammary tumor virus (MuMTV) derived from the milk of of RIII mice has been analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy after treatment with insolubilized trypsin. No alterations were found in viral fine structure by either freeze-etch or negative-stain electron microscopy. No alterations were found in the ability of trypsinized virus to compete in a radioimmune assay for viral antigens. Infectivity experiments indicate no significant differences in the ability of treated virus to infect C57Bl mice. However, significant differences were observed in polypeptide composition. The intensely periodic acid-Schiff-positive band, gp140, was shown by galactose oxidase-borotritide labeling to be degraded into a fragment of 125,000 molecular weight. The major glycoprotein, gp55, was split into fragments of 36,000 and 23,000 molecular weight, both of which stained with periodic acid-Schiff stain. Gp68 was removed from the virus. Experiments with purified, iodinated gp55 showed that the trypsin-induced fragments of gp55 were immunologically active. We conclude that: (i) certain glycoproteins at the surface of MuMTV are accessible to an insoluble form of trypsin, (ii) the trypsin causes a nick in the polypeptide chain without affecting the configuration of the molecule; (iii) the nicked molecules remain bound to the virus; and (iv) the presence of these nicked molecules does not interfere with the biological or antigenic expression of virus function.