Mouse mammary tumor virus and mammary tumorigenesis in wild mice

The current knowledge of the distribution of the mouse mammary tumor virus (MMTV) proviral genomes and the mechanism of mammary tumorigenesis by MMTV in mice, with the main emphasis on Asian feral mice, is reviewed. The relevant earlier discoveries on the mode of MMTV transmission are summarized to provide an outline of the biology of MMTV. Finally, the viral etiology of human breast cancer will be discussed.     

Effect of dexamethasone on expression of endogenous mouse mammary tumor virus sequences in BALB/c tumor cell lines.

BALB/c mammary tumor cell lines which contain only endogenous murine mammary tumor virus (MMTV) sequences respond to dexamethasone (DXS) treatment with minimal (approximately 2-fold) increases in MMTV RNA. This is in marked contrast to the 10? to 20-fold increases observed with cell lines harboring exogenous MMTV variants. Comparison of hybridization results obtained with two complementary DNA probes representative of either the entire MMTV RNA genome or its poly(A)-adjacent sequences suggests that the DXS response of BALB/c lines is also qualitatively different from that of exogenous MMTV-producer cell lines. Thermal stability studies suggested a 2–3% divergence between the RNA sequences of endogenous BALB/c and exogenous C3H viruses, with the 3′-end of the viral RNA appearing to be conserved relative to the rest of the genome.     

Mouse mammary tumor virus production by thymic epithelial cells in vivo

Exogenous mouse mammary tumor viruses (MMTV) replicate in the mammary glands of infected females, and so infect the suckling pups. We have previously shown that the virus is rapidly disseminated to all the lymphoid organs, including the thymus. The present electron microscope immunohistochemical study describes the viral production site in the thymus. Viral buds and viral proteins were restricted to the thymus medullary epithelial cells. MMTV-encoded proteins were identified on the free viral particles and on the budding ones, the ribosomes, the membrane of the endoplasmic reticulum, and on the membrane of the medullary type II epithelial cell vacuolar network. The thymus medullary epithelial cells can thus integrate the virus and allow viral replication. The results support earlier results indicating that in some experimental conditions, epithelial cells may be involved in MMTV-induced negative selection by showing that thymic epithelial cells do express MMTV-encoded proteins.     

Mammary preneoplasia and tumorigenesis in the BALB/c mouse: structure and modification of mouse mammary tumor virus DNA sequences

Mouse mammary tumor virus (MMTV) DNA sequences were examined in mammary tissues from BALB/c mice, including both preneoplastic hyperplastic alveolar nodule (HAN) outgrowth lines, tumors derived from those preneoplastic tissues, and DMBA-induced mammary tumors. Over 60 different HAN and tumors samples were analyzed. The D2 HAN line contained one additional provirus, whereas Cv-2 and Cv-4 HAN lines that are infected with exogenous virus exhibited multiple virus integration events. D2 tumors showed the same provirus pattern as D2 HANs, whereas Cv-2 and Cv-4 tumors exhibited a subset of the acquired proviruses found in the parental HAN populations. Differential methylation patterns of virus-specific sequences were observed that resembled those described for other tissues in which viral DNA replication and integration has occurred, i.e., acquired proviruses were hypomethylated and endogenous proviruses were methylated. In tumors that arose from HAN lines and exhibited only endogenous proviruses, demethylation of the subgenomic Mtv-6 locus was observed. Demethylation of Mtv-6 was not detected in any of the preneoplastic tissues. Altered methylation of Mtv-8 and -9 was observed in both Cv-2 and Cv-4 tumors. Finally, mammary tumors induced by DMBA carried no acquired proviruses and demethylation of endogenous MMTV proviruses was demonstrated.     

Massive mammary gland infection and pregnancy-dependent mammary tumor development in mice infected neonatally with mouse mammary tumor virus (SW) but not in mice infected as adults,despite a dramatic local response

Mouse mammary tumor virus (MMTV) (SW) caused a high incidence (65%) of pregnancy-dependent adenocarcinomas in BALB/c(SW) mice infected as new-borns by suckling their mothers' milk. These tumors were type B adenocarcinomas which developed early, at about 1 year of age. Uninfected breeding females and those infected at an age of 8 weeks by injection of virus had the same low incidence of malignant tumors (13%), and the tumors developed later (at approx. 23–24 months). The low incidence of tumors in adult-infected mice was correlated with partial infection of the mammary glands, and delayed transmission of MMTV(SW) to the offspring. Although the virus was rapidly disseminated in both types of infection, the responses of neonatally infected and adult-infected mice to MMTV(SW) infection and viral superantigen (vSAG) presentation were different. Activation by and presentation of the vSAG was impaired in mice infected neonatally, and tolerance induction by clonal deletion was delayed. Local activation was dramatic in mice infected as adults and clonal deletion followed rapidly. Although interaction between B and T cells is needed for completion of the virus life cycle and viral amplification, the strong local immune response to MMTV(SW) in adult-infected mice limits mammary gland infection, and protects them against mammary tumor development.     

Isolation and characterization of a new mouse mammary tumor virus from BALB/c mice

A novel mouse mammary tumor virus (MMTV) has been isolated from mice of a subline of the BALB/cCrl Med mouse strain designated BALB/cV. Whereas breeding females of the parent BALB/cCrl Med colony have a mammary tumor incidence of 1%, 47% of the breeding females of the BALB/cV subline develop mammary tumors before 10 months of age. Foster nursing experiments demonstrated this virus, termed MMTV (BALB/c), was transmitted only by milk. The novel MMTV isolate was shown to be immunologically related to, but distinct, from the MMTV variants of C3H, GR, and RIII mice by a series of competition radioimmunoassays for the MMTV 28,000-dalton major core protein (p28), and the 52,000 (gp52)- and 36,000-dalton (gp36) major envelope glycoproteins. Monoclonal antibodies directed against MMTV gp36 were also used to clearly distinguish MMTV(BALB/c) from MMTV(C3H), MMTV(RIII), MMTV(GR), MMTV(C3HfC57BL), and MMTV(A). MMTV-specific proviral DNA content of mammary adenocarcinomas arising in the BALB/cV subline was examined with restriction endonucleases and the Southern blot technique, and compared to the MMTV proviral DNA content of BALB/cAnDe mammary tumors. The virus arising from these latter tumors has been termed MMTV(O). Analysis of EcoRI digests of high-molecular-weight DNA from both types of mammary tumors demonstrated additional MMTV-related proviral sequences when compared to the DNA of normal BALB/c tissues. The patterns generated with the restriction endonuclease SacI distinguished the additional MMTV-specific proviral information in the mammary tumors of the BALB/cV mice from the proviral information in tissues containing the GR, C3H, or RIII MMTVs, as well as from the proviral information in the BALB/cAnDe mammary tumors. These immunological and molecular studies thus define MMTV(BALB/c) as a novel MMTV variant.     

Syngeneic mouse mammary carcinoma cell lines: Two closely related cell lines with divergent metastatic behavior

Two cell lines, Met-1^fvb2 and DB-7^fvb2, with different metastatic potential, were derived from mammary carcinomas in FVB/N-Tg(MMTV-PyVmT) and FVB/N-Tg(MMTV-PyVmT ^Y315F/Y322F ) mice, transplanted into syngeneic FVB/N hosts and characterized. The lines maintain a stable morphological and biological phenotype after multiple rounds of in vitro culture and in vivo transplantation. The Met-1^fvb2 line derived from a FVB/N-Tg(MMTV-PyVmT) tumor exhibits invasive growth and 100% metastases when transplanted into the females FVB/N mammary fat pad. The DB-7^fvb2 line derived from the FVB/N-Tg(MMTV-PyVmT ^Y315F/Y322F ) with a “double base” modification at Y315F/Y322F exhibits more rapid growth when transplanted into the mammary fat pad, but a lower rate of metastasis (17%). The Met1^fvb2 cells show high activation of AKT, while DB-7^fvb2 cells show very low levels of AKT activation. The DNA content and gene expression levels of both cell lines are stable over multiple generations. Therefore, these two cell lines provide a stable, reproducible resource for the study of metastasis modulators, AKT molecular pathway interactions, and gene target and marker discovery.     

A Vβ4-specific superantigen encoded by a new exogenous mouse mammary tumor virus

The superantigen (SAg) expressed by mouse mammary tumor virus (MMTV) has been shown to play an essential role in the course of the viral life cycle. In the present study, we describe a Vβ4-specific SAg encoded by a new exogenous MMTV carried by the SIM mouse strain. This is the first report of a viral or bacterial SAg reacting with mouse Vβ4⁺ T cells. Injection of MMTV(SIM) into adult BALB/c mice leads to a rapid and strong stimulation of Vβ4⁺ CD4⁺ T cells, followed by a slow deletion of these cells. Neonatal exposure to the virus also leads to a progressive deletion of Vβ4⁺ T cells. In contrast to other strong MMTV SAg, this new SAg requires the presence of major histocompatibility complex class II I-E molecules to be presented efficiently to T cells. Sequence analysis revealed a new predicted amino acid sequence in the C-terminal polymorphic region of this SAg. Furthermore, sequence comparisons to the most closely related SAg with different Vβ specificities hint at the specific residues involved in the interaction with the T cell receptor.     

Tumor progression and metastasis in murine D2 hyperplastic alveolar nodule mammary tumor cell lines

We have examined tumor progression and metastatic properties of three clonal murine mammary tumor cell lines of recent origin (D2A1, D2.OR and D2.1). These lines were derived from spontaneous mammary tumors which originated from a D2 hyperplastic alveolar nodule (HAN) line. D2A1 cells were more malignant than D2.OR or D2.1 cells, whether measured by experimental metastasis assays after intravenous injection in nude mice or chick embryos,in vivo growth rate of primary tumors following mammary fat pad injection in nude mice, or spontaneous metastasis assay from primary tumors growing in mammary fat pads. D2A1 cells also were more invasivein vitro in a Matrigel invasion assay than D2.1 cells, while the D2.OR cells were non-invasive in this assay. The increased invasiveness and malignancy of D2A1 cells were associated with increased levels of mRNA for the cysteine proteinase cathepsin L. Levels of osteopontin (OPN), nm23, int-1 and int-2 mRNAs were also examined. Nm23 levels were highest in the most malignant cell line. These cell lines provide a model for studying the tumorigenic and metastatic ability of mammary tumor cells and offer several advantages: they were cloned from mammary tumors that originate from a common source of preneoplastic cells (D2HAN); they are of relatively recent origin; and they have spontaneously arrived at different stages of tumor progression.     

Molecular cloning and primary structure analysis of the mouse mammary tumor virus-related element from dwarf hamster genome

Eleven recombinant bacteriophages carrying mouse mammary tumor virus (MMTV)-related sequences (MRS) were isolated from the genomic library of the dwarf hamster (Phodopus sungorus) using radioactively labeled DNA of MMTV as a hybridization probe. There are approximately 50 copies of MRS-Ps per genome, as estimated by the number of positive signals on the autoradiogram of the primary screening plate. MRS of Phodopus sungorus (MRS-Ps) contain regions homologous to the LTR, pol, and, probably, env, but not gag genes of MMTV. A 0.9 kb MRS-Ps fragment was sequenced and proved to be 63% identical to the MMTV pol gene sequence. However, all absolute frames contain multiple stop codons and do not seem to be expressed.     

T cell receptor Vβ repertoire in mice lacking endogenous mouse mammary tumor provirus

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) Vβ segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, ?7, ?8 and ?9) and 38CH (Mtv^?) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, ?7, ?8 or ?9. The TcR Vβ chain (TcR Vβ) usage in these mice was analyzed using monoclonal antibodies specific for TcR Vβ2,Vβ3, Vβ4,β5,Vβ6,Vβ7,Vβ8,Vβ11,Vβ12 and Vβ14 segments. Both Mtv-8⁺ mice and Mtv-9⁺ mice deleted TcR Vβ5⁺ and Vβ11⁺ T cells. Moreover, we also observed the deletion of TcR Vβ12⁺ cells by Mtv-8 and Mtv-9 products. Mtv-6⁺ and Mtv-7⁺ animals deleted TcR Vβ3⁺ and Vβ35⁺ cells, and TcR Vβ6⁺,Vβ7⁺ and Vβ8.1⁺ cells, respectively. Unexpectedly, TcR Vβ8.2⁺ cells were also deleted in some backcross mice expressing Mtv-7. TcR Vβ8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn → Asp) in position 19 on the TcR Vβ8.2 fragment. Reactivities of BALB.D2 TcR Vβ8.2 and 38CH TcR Vβ8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR Vβ2⁺, Vβ4⁺ and Vβ8⁺ CD4⁺ T cell subsets in Mtv-8⁺ and Mtv-9⁺ mice, and TcR Vβ4⁺ CD4⁺ T cells in Mtv-6⁺ and Mtv-7⁺ mice, when compared with the T cell repertoire of Mtv^? mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.     

Differential reactivity of TCR Vβ10 alleles to a mouse mammary tumor virus superantigen

Mouse mammary tumor virus (MMTV) expresses a superantigen (SAg) which plays a critical role in the viral life cycle. We have recently described the new infectious MMTV (SIM) encoding a Vβ4-specific SAg in mice with a TCR-Vβ^b haplotype. We have now compared the SAg activity of this virus in BALB / c mice harboring the TCR-Vβ^a, TCR-Vβ^b or TCR-Vβ^c haplotypes which differ by a central deletion in the TCR-Vβ^a and TCR-Vβ^c locus and by mutations in some of the remaining Vβ elements. Injection of MMTV (SIM) led to a strong stimulation of Vβ4 + CD4 + T cells in TCR-Vβ^b mice, but only to a weak stimulation of these cells in TCR-Vβ^a or TCR-Vβ^c mice. A large increase in the percentage of Vβ10 + cells was observed among CD4 + T cells in mice with the Vβ ^a or Vβ ^c, but not the Vβ ^b TCR-Vβ haplotype. Vβ 10+ cells dominated the response when Vβ10^a/c and Vβ 4 subsets were present together. This is the first report of a viral SAg interacting with murine Vβ10 + cells. Six amino acid differences between Vβ10^{a / c} and Vβ10^b could account for the gain of reactivity of Vβ10^{a / c} to the MMTV(SIM) SAg. No mutations were found in the hypervariable region 4 (HV4) of the TCR. Mutations at positions 22 and 28 introduce into Vβ10^{a / c} the same amino acids which are found at these positions in the MMTV(SIM)-reactive Vβ4. Tridimensional models indicated that these amino acids lie close to HV4 and are likely to be important for the interaction of the SAg with the TCR.