Low incidence of tumourigenicity in agarose colonies from spontaneous murine mammary tumours

In these experiments individual colonies growing in agarose seeded with monocellular suspensions from freshly disaggregated naturally-occurring mouse mammary tumours, induced by the murine mammary tumour virus (MMTV), were reimplanted into mammary fat pads of virus-free mice. It was found that only a small proportion of these colonies generated tumours and that the implantation of multiple colonies in each site did not result in disproportionate, synergistic, increase in tumour takes. It was also observed that the proportion of colonies which were tumourigenic on reimplantation differed for each donor tumour and represented only a small fraction of the total cell population (0.001%-0.1%). However, this value was significantly higher in tumours which produced large numbers of deposits in lung colony assays following i.v. injections, than in tumours of low pulmonary colonisation potential. A point of particular interest was that tumours derived from agarose colonies of spontaneously metastatic donor tumours were substantially more spontaneously metastatic themselves than those from nonmetastatic donors, indicating that this property is heritable through numerous cell divisions, manipulations in vitro and transplantation procedures. From these results it is concluded that measurement of clonogenicity in agar is useful as an index of the capability of a tumour to propagate itself and to colonise new sites, but that individual agarose colonies are not all the progeny of potentially immortal stem cells.     

Tumour incidence, growth, reproduction and longevity in female C3H mice fed polyunsaturated ruminant-derived foodstuffs

Earlier studies with the random-bred Quackenbush mouse strain showed that human-type diets based on linoleic acid-enriched foodstuffs derived from ruminants fed protected polyunsaturated oils have no detrimental effects on growth, reproduction or longevity. Tumour incidence and time of onset of tumour development have now been studied in the inbred, tumour-prone mouse strain C3H, in addition to growth, reproduction and longevity. Mice were fed a polyunsaturated human diet, a conventional human diet, or mouse cubes. The results with C3H mice tended to confirm those with Quackenbush strain mice-growth rates and reproductive productivities were very similar in the two groups eating human diets. Mice on the conventional human diet tended to survive better to about 60 weeks of age than mice on the polyunsaturated diet or the cube diet, after which the mortality rates of the mice on the three diets were similar. The degree of unsaturation of the dietary fat had no significant effect on the incidence of tumours. The tumour incidence was about 40% which, taken in conjunction with the average age of onset (about 80 weeks), suggested that the NIV virus rather than the MMTV virus was responsible. It would appear that the high-fat human diets had no effect on the incidence of mammary tumours caused by this virus.     

Distribution of mouse mammary tumor virus in Asian wild mice.

Several groups of wild mice (Mus musculus) were captured from eight different locations in Asia and bred for several generations in a facility free of any laboratory strains of mice carrying mouse mammary tumor virus (MMTV). The distribution of endogenous MMTV proviral sequences in the liver tissues of these mice was investigated by using Southern blot hybridizations. Four categories of mice were identified. Mice originating from Bogor, Indonesia (Cas-Bgr); He-mei, Taiwan (Cas-Hmi/1); and Malaysia (Cas-Mal) were found to carry an endogenous MMTV provirus consisting of the env, gag-pol, and long terminal repeat sequences. Mice captured from Kojuri, Republic of Korea (Sub-Kjr); Nagoya, Japan (Mol-nag); and three Chinese provinces, Shanghai (Sub-Shh), Beijing (Sub-Bjn), and Jiayuguang (Sub-Jyg/1), appeared to carry defective proviruses. Some mice originating from He-mei (Cas-Hmi/2) and Jiayuguang (Sub-Jyg/2) were found to be completely free of endogenous MMTV. Interestingly, however, the Sub-Jyg/2 mice, after several generations of inbreeding, were found, unlike all of the other subspecies that we examined in the present study, to develop mammary tumors at a high incidence (80 to 90%) with a short period of latency. Electron microscopic examination of the mammary glands and mammary tumors of these mice revealed the presence of numerous intracytoplasmic A, immature, budding, and mature B particles. Furthermore, the mammary tumors were found to contain MMTV proviral sequences. It seems, therefore, that Sub-Jyg/2 mice carry an exogenous MMTV which contributes to their developing mammary tumors.     

C3H/HeN mammary tumor-bearing mice develop type-specific neutralizing antibodies and group-specific precipitating antibodies for the mouse mammary tumor virus.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.     

Modulation of mouse mammary tumor virus production in the MJY-alpha cell line.

Implantation of the mouse mammary tumor virus (MMTV)-producing mammary tumor cell line MJY-alpha into isogeneic mice elicited both humoral and T-cell responses against MMTV virion antigens. The carcinosarcomas which developed from the implanted cells showed a significant decrease in MMTV synthesis, compared with cells remaining in culture, which was detectable as early as 7 days after implantation and for five transplant generations. Electron microscopic examination of thin sections of the tumors revealed that intracytoplasmic A particles, budding particles, and cell-free MMTV B particles were all affected. However, immunofluorescence assays of tumor sections demonstrated the presence of MMTV viral antigens in the cells. Cell cultures initiated from first-, third-, and fourth-generation tumors were morphologically identical to the original in vitro cell line, although virus production was barely detectable. Analysis of the cultures by electron microscopy revealed a significant increase in MMTV virions after in vitro passage 3. Polypeptide profiles obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virions purified from these cultures were identical to MMTV. Immunodiffusion demonstrated the cross-reactivity between these virions and MMTV particles obtained from mouse milk. In vitro treatment of MJY-alpha cell cultures with rabbit anti-MMTV antiserum resulted in a reduction of extracellular MMTV virions, as well as alterations in their sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide patterns.     

Coexpression of exogenous and endogenous mouse mammary tumor virus RNA in vivo results in viral recombination and broadens the virus host range.

Mouse mammary tumor virus is a replication-competent B-type murine retrovirus responsible for mammary gland tumorigenesis in some strains of laboratory mice. Mouse mammary tumor virus is transmitted horizontally through the milk (exogenous or milk-borne virus) to susceptible offspring or vertically through the germ line (endogenous provirus). Exogenously acquired and some endogenous mouse mammary tumor viruses are expressed at high levels in lactating mammary glands. We show here that there is packaging of the endogenous Mtv-1 virus, which is expressed at high levels in the lactating mammary glands of C3H/HeN mice, by the virions of exogenous C3H mouse mammary tumor virus [MMTV(C3H)]. The mammary tumors induced in C3H/HeN mice infected with exogenous MMTV (C3H) virus contained integrated copies of recombinant virus containing a region of the env gene from an endogenous virus. This finding indicates that there was copackaging of the Mtv-1 and MMTV(C3H) RNAs in the same virions. Moreover, because Mtv-1 encodes a superantigen protein with a V beta specificity different from that encoded by the exogenous virus, the packaging of Mtv-1 results in an infectious virus with a broader host range than MMTV(C3H).     

Chemical carcinogen-mouse mammary tumor virus interactions in cell transformation

We have studied the process of mammary cell transformation in vitro using a single cell clone (Clone 18) from a presumptive epithelial cell line, C57MG, derived from a normal mammary gland; a mouse mammary tumor virus (MMTV) host-range variant (RIII)vp4; and the potent initiating carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). After several serial subcultures, cells treated with virus and then with carcinogen exhibited an altered (transformed) morphology, a dramatic increase in anchorage independence, an increase in multinucleation after exposure to cytochalasin B, an enhanced ability to proliferate in low Ca2+ (0.01 mM) medium, and tumorigenicity when inoculated subcutaneously into athymic (nude) mice. Although some of these phenotypic alterations were observed also in cultures treated singly with MMTV or DMBA and in cultures exposed to DMBA before infection with MMTV, enhanced cytochalasin B multinucleation and tumorigenicity were properties observed only in mass cultures of cloned cells first infected with MMTV and then exposed to DMBA. This demonstrates for the first time that exposure of presumptive mammary epithelial cells to MMTV followed by DMBA, but not to either agent alone or to DMBA followed by MMTV, results in malignant transformation of these cells.     

Incomplete proviral genome of mouse mammary tumour virus is present on chromosome Y of NIH Swiss and some genetically related mouse strains.

An incomplete proviral genome of endogenous mammary tumour virus (MMTV) was found in DNA of several strains of mice. This MMTV-related sequence was assigned to the Y chromosome since it was clearly observed in male mice only. This MMTV provirus contained a sequence related to LTR (long terminal repeat), but not to gag-pol and env genes. NFS, NIH Swiss/S, STS/A, and DD/Tbr mice have this sequence but BALB/cHeA, SHN, SLN, C57BL/6NJcl, C3H/HeNJcl and CBA/JJcl mice are negative. In the strains containing this sequence, a DNA test for the sequence makes it possible to easily distinguish the DNAs of male or female mice.     

Neoplastic response of turkey liver to MC29 leukosis virus.

Twelve to fourteen days after intravenous inoculation of MC29 virus (1 x 10(5)LD50) into 1-day-old turkeys, liver tumour developed in 100% of the infected animals and led to death of birds. The tumour proved to be hepatomas histologically and electron microscopically. Numerous C-type particles were detectable among the tumour cells, as well as budding from the cell membrane. C-type particles were also observed in the tumour-free liver tissue in the spaces between the cells, budding was not detectable. Large number of virus particles were found in spleen extracellularly and intracellular vacuoles. Kidney tumours did not develop, but a few extracellular virus particles were located in the tissue. The MC29 virus-induced primary liver tumour in the turkey seems to be a suitable model for the morphological study of the relationship between oncogenic viruses and eukaryotic cells.     

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.     

Both T and B cells shed infectious mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) infected both B and T tissue culture cells and primary B and T cells in vivo after milk-borne transmission of the virus. The infected tissue culture cells processed viral proteins, and both these and primary B and T cells shed virus when cultured in vitro. Moreover, the infected B and T tissue culture cells transmitted virus to uninfected mammary gland cells in vitro. The level of infection of these different cell types in vivo was dependent on the strain of mouse, with C3H/HeN mice showing greater B-cell infection and BALB/c mice greater T-cell infection after nursing on MMTV-infected C3H/HeN mothers. Although their B cells were less infected, BALB/c mice developed tumors more rapidly than C3H/HeN mice. These results indicate that both infected T and B cells are potential carriers of MMTV in vivo.     

Initial stages of mammary tumor virus infection are superantigen independent

Exogenous mouse mammary tumor virus (MMTV) is transmitted via the milk from infected mothers to newborn pups. Efficient MMTV transmission is dependent on proliferation of T cells with particular TCR beta-chains, which occurs upon recognition of virally encoded superantigen (SAg) bound to MHC class II molecules. It is assumed that infection of these dividing cells favors MMTV amplification. SAg is important for MMTV infection, as mice that lack SAg-cognate T cells due to expression of endogenous Mtv loci or mice that express inappropriate MHC haplotypes unable to present viral SAg efficiently were shown to be resistant to MMTV infection. However, this resistance was not absolute, as these mice developed late onset MMTV-induced mammary tumors. In this study, we show that the success of initial MMTV infection in neonates is independent of SAg function but depends on the developmentally regulated proliferation of target cells. However, SAg was absolutely required for virus spread following completion of this proliferative stage.     

Proviruses of mouse mammary tumor virus in normal and neoplastic tissues from GR and C3Hf mouse strains.

We analyzed two experimental situations to assess the role of endogenous mouse mammary tumor virus (MMTV) DNA in the genesis of mammary carcinomas. (i) GR mice carry in their germ line one or more proviruses indistinguishable by limited restriction mapping from the proviruses introduced into cells by experimental infection with the highly tumorigenic virus isolated from GR mouse milk, MMTV(GR). Most tumors arising in GR mice contain one or more proviruses at various sites in tumor DNA in addition to those present endogenously. Detection of these new proviruses is possible as a consequence of the clonal or quasiclonal character of the tumors. (ii) C3H/He mice carry three units of endogenous viral DNA, none of which resembles the DNA of the commonly encountered strains of milk-borne MMTV. Nevertheless, MMTV-associated tumors arise late in life when these animals are removed from the influence of milk-borne virus; the responsible agent, MMTV(C3Hf), can also produce tumors in BALB/c mice. We found that tumors arising in both C3Hf/He mice and BALB/c mice infected with MMTV(C3Hf) were clonal or quasiclonal and contained one or more new copies of proviral DNA at various sites in the host genome. These new proviruses were readily distinguished from the proviruses of the common milk-borne virus strains and closely resembled unit II of endogenous MMTV DNA (Cohen et al., J. Virol., 32:483-496). Thus, in both experimental systems, we found evidence for new proviruses in mammary tumors, despite the preexistence of similar or identical proviruses in the germ line. The results suggest that the repositioning of MMTV proviruses may be required for the full expression of the oncogenic potential of endogenous MMTV DNA.     

Chemical carcinogen-mouse mammary tumor virus interactions in cell transformation

Summary We have studied the process of mammary cell transformation in vitro using a single cell clone (Clone 18) from a presumptive epithelial cell line, C57MG, derived from a normal mammary gland; a mouse mammary tumor virus (MMTV) host-range variant (RIII)vp4; and the potent initiating carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). After several serial subcultures, cells treated with virus and then with carcinogen exhibited an altered (transformed) morphology, a dramatic increase in anchorage independence, an increase in multinucleation after exposure to cytochalasin B, an enhanced ability to proliferate in low Ca²⁺ (0.01 mM) medium, and tumorigenicity when inoculated subcutaneously into athymic (nude) mice. Although some of these phenotypic alterations were observed also in cultures treated singly with MMTV or DMBA and in cultures exposed to DMBA before infection with MMTV, enhanced cytochalasin B multinucleation and tumorigenicity were properties observed only in mass cultures of cloned cells first infected with MMTV and then exposed to DMBA. This demonstrates for the first time that exposure of presumptive mammary epithelial cells to MMTV followed by DMBA, but not to either agent alone or to DMBA followed by MMTV, results in malignant transformation of these cells. Support for these studies was provided in part by the National Cancer Institute, Bethesda, Md., Contract N01-CP-01018.     

Mouse mammary tumor virus proviral sequences congenital to C3H/Sm mice are differentially hypomethylated in chemically induced, virus-induced, and spontaneous mammary tumors

C3H/Sm mice have lost the exogenous milk-borne mouse mammary tumor virus (MMTV) characteristic of the C3H strain and have a very low (1.5%) incidence of spontaneous mammary tumors, yet they are highly susceptible to mammary carcinogenesis by either chemical carcinogens or infection with the milk-borne virus. We have analyzed the MMTV proviral DNA content of normal tissues and of spontaneous, virus-induced, and chemically induced mammary tumors by restriction endonuclease digestion and Southern blot analysis. Although the results clearly showed additional MMTV sequences in the virus-induced tumor which are not present in normal liver DNA, none of the spontaneous or chemically induced tumors could be shown to contain either newly acquired exogenous or amplified endogenous MMTV sequences. Interestingly, mammary tumors arising in C3H/Sm mice treated simultaneously with infectious MMTV (C3H) and dimethylbenz[a]anthracene (DMBA) possessed new exogenous MMTV DNA even though no quantitative change in tumor production was observed when these mice were compared with C3H/Sm mice treated with DMBA alone (Smith et al., Int. J. Cancer 26:373-379, 1980). Our data indicate that the endogenous MMTV proviral units are extensively methylated in normal tissues, such as livers and normal nonlactating mammary glands. In the absence of MMTV (C3H), we found that in the rare, spontaneously occurring C3H/Sm mammary tumors, certain endogenous MMTV sequences were specifically hypomethylated. Hypomethylation of endogenous MMTV sequences was also noted in the chemically induced mammary tumors, even though radioimmune competition assays for MMTV gp52 and p28 are negative (Smith et al., Int. J. Cancer 27:81-86, 1981). Our results support the conclusion that amplification of endogenous MMTV sequences is not intrinsic to C3H/Sm mouse mammary tumors arising spontaneously or after induction by chemicals. On the other hand, integration of exogenous MMTV DNA into the genome was a constant feature of mammary tumors developing in MMTV (C3H)-infected C3H/Sm mice, even when DMBA was used as the carcinogen. Hypomethylation of some endogenous MMTV sequences is characteristic of C3H/Sm mammary tumors, whether spontaneous or induced by chemicals, which suggests that these sequences are located in actively transcribing regions of the tumor cell genome.     

Exogenous mouse mammary tumor virus proviral DNA isolated from a kidney adenocarcinoma cell line contains alterations in the U3 region of the long terminal repeat.

Mouse mammary tumor virus (MMTV) is a B-type retrovirus which induces predominantly mammary carcinomas after a relatively long latency period. To date, very little is known about the reasons for the strict tissue specificity of MMTV. The BALB/cf/Cd strain of mice, which was infected with milk-borne MMTV (C3H), shows a high incidence of kidney adenocarcinomas, and our data suggest that MMTV might be involved in the formation of these tumors. Newly integrated exogenous MMTV proviruses were found in the genome of transplanted tumor cells as well as in the DNA of a cell line derived from one tumor, but not in normal cells of BALB/cf/Cd mice. The MMTV DNA in these tumor cells was transcribed and viral RNA synthesis was strongly stimulated by glucocorticoid hormones. Viral structural polypeptides, comparable in size and antigenicity to MMTV polypeptides of infected mammary tumor cells were synthesized and processed normally in the cell line and were organized correctly into intracytoplasmic particles. Heteroduplex analysis of the molecularly cloned MMTV proviral DNAs of kidney and mammary tumor origin revealed a high degree of homology in the gag, pol, and env genes. A striking difference, however, was observed in the U3 region of the two LTRs that might relate to the different tissue specificity of the two viruses.     

Critical role of dendritic cells in mouse mammary tumor virus in vivo infection

Mouse mammary tumor virus (MMTV) is a milk-transmitted betaretrovirus that causes mammary tumors in mice. Although mammary epithelial cells are the ultimate targets of MMTV, the virus utilizes components of the host immune system to establish infection. Previous studies indicated that dendritic cells play a role in MMTV infection. Here we show that dendritic cells are the first cells to be infected by MMTV in vivo and that they are capable of producing infectious virus that can be transmitted to other cell types. Moreover, upon contact with the virus, dendritic cells became more mature and migrated in response to the chemokine macrophage inflammatory protein 3beta. Finally, we demonstrate that targeted ablation of dendritic cells in vivo dramatically attenuated MMTV infection. These data indicate that MMTV infection of dendritic cells is critical to initial propagation of the virus in vivo.