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.     

Hormonal regulation of mouse mammary tumor growth

In view of reports that human breast cancer cells secrete growth factors that can replace estradiol in sustaining tumor growth [1], we have investigated whether hormone independent (HI) GR mouse mammary tumors can sustain growth of estrogen-depleted hormone dependent (HD) tumors. HD GR mammary tumor TSl 106 was grafted subcutaneously in the right flank of estrone plus progesterone treated castrated (020 X GR)F1 mice. After 2 weeks the estrone treatment was stopped and the mice received 50, 100 or 150 mg HI GR mammary tumor TSl 104 in the left flank. However, the regression of the HD tumor due to estrone depletion was not prevented or retarded by the HI grafts. In other experiments we investigated integrations of mouse mammary tumor virus (MMTV) proviral DNA in the DNA of GR mammary tumors. We could demonstrate the presence of two cell populations in tumor TSl 96, both HD but differing in MMTV DNA integration events. Our data indicate that exogenous integrations of MMTV proviruses can take place in mouse mammary tumor DNA without loss of hormone dependency of the tumors. Like in GR/Mtv-2+ mice, mammary tumor transplants differing in MMTV proviral integrations are also observed in 020/Mtv-2+ mice.     

A novel mechanism of resistance to mouse mammary tumor virus infection

Exogenous mouse mammary tumor virus (MMTV) is carried from the gut of suckling pups to the mammary glands by lymphocytes and induces mammary gland tumors. MMTV-induced tumor incidence in inbred mice of different strains ranges from 0 to as high as 100%. For example, mice of the C3H/HeN strain are highly susceptible, whereas mice of the I/LnJ strain are highly resistant. Of the different factors that together determine the susceptibility of mice to development of MMTV-induced mammary tumors, genetic elements play a major role, although very few genes that determine a susceptibility-resistance phenotype have been identified so far. Our data indicate that MMTV fails to infect mammary glands in I/LnJ mice foster nursed on viremic C3H/HeN females, even though the I/LnJ mammary tissue is not refractory to MMTV infection. Lymphocytes from fostered I/LnJ mice contained integrated MMTV proviruses and shed virus but failed to establish infection in the mammary glands of susceptible syngeneic (I x C3H.JK)F(1) females. Based on the susceptible-resistant phenotype distribution in N(2) females, both MMTV mammary gland infection and mammary gland tumor development in I/LnJ mice are controlled by a single locus.     

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.     

Genetics of mouse mammary tumor virus-induced mammary tumors: linkage of tumor induction to the gag gene

Retroviruses are believed to induce tumors by acting as insertional mutagens that activate expression of cellular protooncogenes. Indeed, almost 90% of mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/He mice show upregulation of Int protooncogenes. We have analyzed three different MMTV variants [MMTV(C3H), MMTV(HeJ), and a genetically engineered MMTV hybrid provirus (HP)] for tumorigenicity in mice from two distinct genetic backgrounds. All three viruses were tumor causing in BALB/cJ mice. However, only MMTV(C3H), but not MMTV(HeJ) or HP, induced mammary tumors in C3H/He mice. All of the viruses were infectious on either background and up-regulated expression of Int genes in tumors they induced. Like HP, MMTV(HeJ) was found to be a genetic recombinant between endogenous Mtv1 provirus and exogenous MMTV(C3H). Sequence comparison of MMTV variants linked the tumorigenicity of MMTV(C3H) to the gag region of the retrovirus.     

Host genetic background effect on the frequency of mouse mammary tumor virus-induced rearrangements of the int-1 and int-2 loci in mouse mammary tumors.

The frequency with which int-1 and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75% of C3H mammary tumors, int-1 is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P less than 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the R111 mouse strain (59%) occurred at a significantly (P less than 0.025) higher frequency than in BALB/cfR111 (25%) mammary tumors. Moreover, in BALB/cfR111 mammary tumors, there is evidence that rearrangement of int-1 and int-2 does not occur independently (P less than 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and R111 mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV.     

Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene

We have used transgenic mice that carry an activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV) promoter to assess the stepwise progression of carcinogenesis in mammary epithelium. Unlike the stochastic occurrence of solitary mammary tumors in transgenic mice bearing the MMTV/c-myc or the MMTV/v-Ha-ras oncogenes, transgenic mice uniformly expressing the MMTV/c-neu gene develop mammary adenocarcinomas that involve the entire epithelium in each gland. Because these tumors arise synchronously and are polyclonal in origin, expression of the activated c-neu oncogene appears to be sufficient to induce malignant transformation in this tissue in a single step. In contrast, expression of the c-neu transgene in the parotid gland or epididymis leads to benign, bilateral epithelial hypertrophy and hyperplasia which does not progress to full malignant transformation during the observation period. These results indicate that the combination of activated oncogene and tissue context are major determinants of malignant progression and that expression of the activated form of c-neu in the mammary epithelium has particularly deleterious consequences.     

Generation of a tumorigenic milk-borne mouse mammary tumor virus by recombination between endogenous and exogenous viruses.

Two novel exogenous mouse mammary tumor viruses (MMTV), BALB2 and BALB14, that encode superantigens (Sags) with Vbeta2+ and Vbeta14+ specificities, respectively, were found in the BALB/cT mouse strain. BALB/cT females were crossed with AKR/J males to generate F1 females. Foster nursing of BALB/cT mice on (BALB/cT x AKR/J)F1 mothers resulted in the generation of a new mouse strain, BALB/cLA, that had acquired a new exogenous MMTV (hereafter called LA) with a Vbeta6+/Vbeta8.1+-T-cell-specific Sag. Sequence analysis of the long terminal repeats of the BALB2, BALB14, and LA viruses indicated that LA virus resulted from recombination between BALB14 and the endogenous Mtv-7 provirus. Mtv-7 is expressed only in lymphoid tissues but not the mammary glands of Mtv-7-containing mouse strains such as AKR. In contrast, LA virus was highly expressed in the mammary gland, although it had the sag-specific region from Mtv-7. The LA virus, as well as different recombinant viruses expressed in the mammary glands of (BALB/cT x AKR/J)F1 mice, acquired a specific DNA sequence from BALB14 virus that is required for the mammary-gland-specific expression of MMTV. Since the Sag encoded by LA virus strongly stimulated cognate T cells in vivo, selection for recombinant virus with the Mtv-7 sag most likely occurred because the increased T-cell proliferation resulted in greater lymphoid and mammary gland cell infection. As a result of the higher virus titer, 80% of BALB/cLA females developed mammary gland tumors, although the incidence was only 40% in BALB/cT mice.     

Androgen regulation by the long terminal repeat of mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) has long been implicated in mouse mammary carcinogenesis, and it is now well established that the long terminal repeat (LTR) contains regulatory sequences responsible for glucocorticoid-mediated induction of viral RNA. However, we have demonstrated previously that androgens as well as glucocorticoids can regulate MMTV RNA in the S115 mouse mammary tumor cell line. To determine if androgens act directly on the LTR in these cells, plasmids were constructed with the MMTV LTR joined to the coding sequences of genes not normally expressed in the cells. Following transfection of these chimeric genes into S115 cells, we show that the expression of the genes is regulated by both androgens and glucocorticoids. Furthermore, hormonal regulation is also conferred by the LTR on the neighboring guanine phosphoribosyltransferase (gpt) gene. Thus, androgens can act on the LTR of MMTV when the appropriate receptors are present in the cells, and this interaction can influence the expression of additional adjacent genes.     

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.     

Expression pattern of mouse mammary tumor virus in transgenic mice carrying exogenous proviruses of different origins.

To study the tissue specificity of mouse mammary tumor virus (MMTV) gene expression, we developed two series of transgenic mice, containing the MMTV proviral DNA of mammary (GR) and kidney (C3H-K) origin. The expression pattern in the MMTV(GR) transgenic mice is very similar to that observed in infected animals, e.g., a strong preference for viral expression in the lactating mammary glands and lower levels of expression in salivary glands, lymphoid tissues, and male reproductive organs. One line of transgenic mice carrying the C3H-K provirus has a similar expression pattern, indicating that MMTV(C3H-K), despite a striking alteration in the U3 region of its long terminal repeat, can be expressed in the same tissues as the wild-type MMTV.     

Tumorigenesis by mouse mammary tumor virus: Evidence for a common region for provirus integration in mammary tumors

We have prepared specific probes for unique-sequence cellular DNA adjacent to each of the newly integrated proviruses in tumors induced by mouse mammary tumor virus (MMTV). The use of such probes to screen a large number of independent mammary tumors in the BR6 strain of mouse has indicated that in at least 17 out of the 40 tumors examined so far, an MMTV provirus has integrated into a common chromosomal domain. A 10 kb Eco RI fragment of single copy DNA from this region has been isolated and partially characterized by restriction enzyme mapping. Of the proviruses located within this fragment in different tumors, all but one are complete, in the same orientation, and clustered within about 3 kb of cellular DNA. These findings are consistent with an insertional mutagenesis model for tumorigenesis by MMTV, in which the integration of a provirus in a particular region of cellular DNA may activate a neighboring oncogene. The region we describe here appears to be different from that reported for mammary tumors in the C3H strain of mouse.     

Isolation of the mouse mammary tumor virus sequences not transmitted as germinal provirus in the C3H and RIII mouse strains.

Radioactive 60-70S RNA from the mouse mammary tumor virus (MMTV) produced by the C3H mouse mammary tumor cell line (Mm5mt) hybridized to a greater extent, and at a lower Cot1/2 value, to the DNA of C3H mammary tumor cells than to the DNA of C3H liver cells. The 125I-labeled MMTV (C3H) 60-40S RNA was annealed to a vast excess of DNA from C3H livers, and single-stranded RNA was eluted from hydroxylapatite and recovered. This "recycled RNA" did not hybridize to the DNA of the apparently normal organs tested from normal or from mammary tumor-bearing C3H mice, but hybridized extensively to both the DNA from the C3H mammary tumor cell line and the DNA from spontaneous C3H mammary tumors. This hybridization could be competed out by the addition of unlabeled MMTV 60-70S RNA but was unaffected by the addition of unlabeled 60-70S RNA of C3H type C virus. Similar experiments were conducted with the RIII mouse strain. We therefore report on the isolation of the sequences of the RNA genomes of the MMTVs from C3H and RIII mice that are transmitted by some mechanism other than via the germ line. These studies further define the differences, via molecular hybridization, between the MMTV-S and the MMTV-L in both C3H and RIII mice.     

Integration of the DNA of mouse mammary tumor virus in virus-infected normal and neoplastic tissue of the mouse.

We have used restriction endonucleases which cleave the DNA of mouse mammary tumor virus (MMTV) at one site (Eco RI) and several sites (Pst I, Sac I and Bam HI) to study infection and mammary tumorigenesis in mice. Proviruses acquired during infection of BALB/c mice foster-nursed by virus-producing C3H females can be distinguished from the MMTV proviruses endogenous to uninfected BALB/c mice by the nature of the fragments generated with Pst I and Bam HI. Using this assay, we show that lactating mammary glands as well as mammary tumors from BALB/cfC3H mice have acquired MMTV DNA, and that a minimum of approximately 10% of normal glandular cells can be infected. The new proviruses appear to be linked to cellular DNA of mammary tumors and infected lactating mammary glands within a limited region (0.2 x 10(6) daltons) of the viral DNA; the location of this region, based upon mapping studies with unintegrated MMTV DNA, suggests that the orientation of these proviruses is colinear with linear DNA synthesized in infected cells and thus approximately colinear with the viral RNA. Comparisons of many mammary tumors and studies of lactating mammary glands with a high proportion of independently infected cells indicate that a large number of sites in the cellular genome can accommodate a new provirus; the acquired proviruses are rarely, if ever, found in tandem with each other or with endogenous proviruses. We cannot, however, distinguish between random integration and integration into a large number of preferred sites in the host genome. Since Eco RI and Bam HI cleavage of DNA from each mammary tumor generates a unique set of viral-specific fragments, we propose that the tumors are composed principally of cells derived from a subset of the many infected cells in a mammary gland; this proposal is supported by our finding that Eco RI digestion of DNA from several transplants of a primary tumor yields the pattern characteristic of the primary tumor.     

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.