Prevalence of serologic reactivity against four strains of mouse mammary tumour virus among US women with breast cancer.

Mouse mammary tumour virus (MMTV) causes breast cancer in mice, and MMTV-specific antibodies develop to high titers among mice infected as adults. Whether MMTV or a related virus infects humans is uncertain, because MMTV DNA sequences have been detected inconsistently and because serologic methods have varied widely. The current study used immunoblot and immunoprecipitation with four strains of MMTV (RIII, FM, C3H, and LA) to detect specific antibodies in 92 sera from US women with breast cancer and in masked dilutions of monoclonal hybridoma and hyperimmunised goat positive-control reagents. In these positive controls, MMTV antibodies of the expected molecular weights were detected at high titer (1 : 100 in the monoclonal reagent, 1 : 10000 in the hyperimmunised goat serum). Nearly 30% of the sera from women with breast cancer had at least one faint band on an immunoblot, but none of these matched the molecular weight of bands revealed by probing the same blot strips with the goat serum. The goat serum readily immunoprecipitated MMTV antigens from all four strains of MMTV, but MMTV antigens were not immunoprecipitated by any of the six breast cancer sera that had four or more nonspecific immunoblot bands. Thus, among women with breast cancer, we found no MMTV-specific antibodies. The upper 95% confidence limit implies that MMTV seroprevalence among breast cancer patients does not exceed 3%.

Cellular ITAM-containing proteins are oncoproteins in nonhematopoietic cells.

Immunoreceptor tyrosine-based activation motifs (ITAMs) are involved in the transduction of signals necessary for activation, differentiation, and survival in hematopoietic cells. Several viruses have been shown to encode ITAM-containing transmembrane proteins. Although expression of these viral proteins has in some cases been shown to transform nonhematopoietic cells, a causal role for a functional ITAM in this process has not been elucidated. To examine the potential transforming properties of ITAM-containing proteins, a recombinant protein consisting of ITAM-containing cytoplasmic regions of the B-cell antigen receptor was expressed in immortalized murine mammary epithelial and fibroblast cells. Mammary epithelial cells expressing this construct exhibited depolarized morphology in three-dimensional cultures. This transformed phenotype was characterized by a loss of anchorage dependence and hallmarks of epithelial to mesenchymal transition. Fibroblasts expressing this ITAM construct also lost contact inhibition and anchorage dependence. The transformed phenotype seen in both cell types was abrogated upon tyrosine to phenylalanine substitutions of the ITAMs. Inhibition of Syk tyrosine kinase, which associates with the ITAM, also prevented cell transformation. Our results indicate that expression of a nonviral ITAM-containing protein is sufficient for cell transformation. Despite lacking intrinsic enzymatic activity, ITAM-containing proteins can function as potent oncoproteins by scaffolding downstream mediators.

Interleukin-4 up-regulates mouse mammary tumor virus expression yet is not required for in vivo virus spread.

The mouse mammary tumor virus (MMTV) superantigen induces T-cell production of cytokines, such as interleukin-4, which in turn increase MMTV transcription. However, interleukin-4 is not required for in vivo virus spread, because mice lacking interleukin-4 or the STAT6 transcription factor showed wild-type infection of lymphoid and mammary tissue. In spite of this, mammary tumor incidence was decreased in STAT6 null mice.

Promoter function of the angiogenic inducer Cyr61gene in transgenic mice: tissue specificity, inducibility during wound healing, and role of the serum response element.

The cysteine-rich angiogenic protein 61 (Cyr61) is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, stimulates cell migration, and enhances growth factor-induced cell proliferation. Cyr61 also promotes chondrogenic differentiation and induces neovascularization. In this study, we show that a 2-kb fragment of the Cyr61 promoter, which confers growth factor-inducible expression in cultured fibroblasts, is able to drive accurate expression of the reporter gene lacZ in transgenic mice. Thus, transgene expression was observed in the developing placenta and embryonic cardiovascular, skeletal, and central and peripheral nervous systems. The sites of transgene expression are consistent with those observed of the endogenous Cyr61 gene as determined by in situ hybridization and immunohistochemistry. The transgene expression in the cardiovascular system does not require the serum response element, a promoter sequence essential for transcriptional activation of Cyr61 by serum growth factors in cultured fibroblasts. Because the serum response element contains the CArG box, a sequence element implicated in cardiovascular-specific gene expression, the nonessential nature of this sequence for cardiovascular expression of Cyr61 is unexpected. Furthermore, the Cyr61 promoter-driven lacZ expression is inducible in granulation tissue during wound healing, as is synthesis of the endogenous Cyr61 protein, suggesting a role for Cyr61 in wound healing. Consistent with this finding, purified Cyr61 protein promotes the healing of a wounded fibroblast monolayer in culture. In addition, we mapped the mouse Cyr61 gene to the distal region of chromosome 3. Together, these results define the functional Cyr61 promoter in vivo, and suggest a role of Cyr61 in wound healing through its demonstrated angiogenic activities upon endothelial cells and its chemotactic and growth promoting activities upon fibroblasts.

Using genetics to probe host-virus interactions; the mouse mammary tumor virus model.

It is clear that there is genetic variation among different individuals in their susceptibility to infection by viruses and other pathogens. Identification of the genes involved in conferring resistance or susceptibility to viral infection will allow us to understand both mechanisms of infection and pathogenesis and to develop reagents for treating or preventing them. Because of the large number of genetically well-characterized inbred mouse strains and the ability to generate targeted germ line mutations, this species is particularly well-suited for such analysis. This review focuses on how the use of genetics to study the retrovirus mouse mammary tumor virus allowed the dissection of both the viral infection pathway and the response of the host to this infection.

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.

Expression of mouse mammary tumor virus envelope protein does not prevent superinfection in vivo or in vitro.

Inbred mice expressing endogenous mouse mammary tumor virus envelope proteins can be infected with exogenous virus, and the mammary tumors that develop in these mice usually have many proviruses integrated in their genomes, indicating that this virus is not subject to receptor interference. We show here that transgenic mice expressing an exogenous mouse mammary tumor virus (C3H) envelope protein can still be infected with this virus. Moreover, cultured mammary gland cells expressing the mouse mammary tumor virus (C3H) envelope protein can be superinfected with pseudotyped viruses bearing that same protein. Thus cellular expression of the mouse mammary tumor virus envelope protein does not block superinfection in vivo or in vitro.

Mammary gland expression of mouse mammary tumor virus is regulated by a novel element in the long terminal repeat.

Mouse mammary tumor virus (MMTV) infects both lymphoid tissue and lactating mammary gland during its infectious cycle, but some endogenous MMTVs are transcribed only in lymphoid cells. We found a lymphoid cell-specific endogenous MMTV that was converted to a milk-borne, infectious virus through recombination with an exogenously transmitted MMTV. The changed expression pattern correlated with the alteration of a single base pair in the long terminal repeat of the lymphoid cell-specific virus. Transgenic mice with the element from either the milk-borne or lymphoid cell-specific virus upstream of the chloramphenicol acetyltransferase reporter gene showed the same pattern of expression as the virus from which the regulatory sequences were derived. Electrophoretic mobility shift assays with mammary cell extracts showed that the site from the milk-borne virus was preferentially bound by a prolactin-inducible factor that poorly bound the altered site from the lymphoid cell-specific virus. The complex that formed on the milk-borne virus-specific oligonucleotide supershifted with anti-Stat5b antibody. Mice lacking either Stat5a or Stat5b had dramatically reduced levels of MMTV transcripts in mammary gland but not in lymphoid tissue. Thus, a member of the STAT family of transcription factors is involved in the tissue-specific expression of mouse mammary tumor virus in vivo. This is the first example of the involvement of a member of the STAT family of transcription factors in the control of tissue-specific expression.

B and T cells are required for mouse mammary tumor virus spread within the mammary gland.

Mouse mammary tumor virus (MMTV) is an infectious retrovirus transmitted through milk from mother to newborns. MMTV encodes a superantigen (SAg) whose activity is indispensable for the virus life cycle, since a genetically engineered virus with a mutation in the sag gene neither amplified in cells of the immune system of suckling pups nor infected their mammary glands. When wild-type MMTV was injected directly into the mammary glands of uninfected pubescent mice, their lymphoid as well as mammary gland cells became virus infected. To test whether this infection of lymphoid cells was dependent on SAg activity and required for virus spread within the mammary gland, we performed mammary gland injections of wild-type MMTV(C3H) into two strains of transgenic mice that lacked SAg-cognate, V beta 14+ T cells. Neither the MTV-ORF or LEL strains showed infection of their mammary glands. Moreover, no MMTV infection of their peripheral lymphocytes was detected. Similar experiments with mice lacking B cells (mu-chain knockouts) showed no detectable virus spread in the mammary glands or lymphoid tissues. These data suggest that SAg activity and MMTV-infected lymphocytes are required, not only for initial steps of viral infection, but also for virus spread within the mammary gland. Virus spread at late times in infection determines whether MMTV induces mammary tumors.

A novel membrane protein is a mouse mammary tumor virus receptor.

Mouse mammary tumor virus (MMTV) infects a number of different cell types, including mammary gland and lymphoid cells, in vivo. To identify the cellular receptor for this virus, a mouse cDNA expression library was transfected into Cos-7 monkey kidney cells, and those transfected cells able to bind virus were selected by using antibody against the virus's cell surface envelope protein, gp52. One clone isolated from a library prepared from newborn thymus RNA, called MTVR, was able to confer virus binding to both monkey and human cells; this binding was blocked by anti-MTVR antibody. Moreover, transfection of MTVR into CV1 cells rendered them susceptible to infection by a murine leukemia virus-based retrovirus vector pseudotyped with the MMTV envelope protein. An epitope-tagged MTVR cofractionated with cellular membranes. Coimmunoprecipitation of the MMTV envelope protein and a MTVR-rabbit Fc fusion protein showed that these two proteins bound to each other. The MTVR sequence clone is unique, shows no homology to known membrane proteins, and is transcribed in many tissues.

Mouse mammary tumor virus and its interaction with the immune system.

Mouse mammary tumor virus (MMTV) is a retrovirus that is transmitted through milk to offspring. Gut-associated B cells are the first cells to be infected during virus transmission, and these cells present a virus-encoded superantigen to cognate T cells. This allows MMTV to replicate and amplify in activated lymphocytes and ultimately results in virus transmission to the mammary epithelial cells. Because the superantigen has profound effects on the T cell repertoire and because MMTV replicates in lymphoid cells, loss of immune response to the virus may also play a role in its ability to persist within its host. Transcriptional control of MMTV expression also plays an important part in this pathway and DNA recognition sequences for transcription factors that allow its expression in lymphoid organs and mammary epithelia are encoded within the virus. Thus, this virus has evolved to take maximum advantage of its host's biology.

The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression.

The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.

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.

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.

Mouse mammary tumor virus: a virus that exploits the immune system.

Mouse mammary tumor virus (MMTV) causes mammary carcinomas and T-cell tumors in mice. MMTV variants that induce T-cell tumors have a large deletion within the U3 region of the long terminal repeat (LTR) compared to MMTV strains that induce mammary tumors. We provide evidence here that T-cell tropic MMTV strains lack a redundant binding site for a cellular protein called NBP (negative regulatory element binding protein). The lack of NBP-binding sites in T-cell tropic MMTV strains presumably leads to higher levels of transcription in T-cells during the MMTV life cycle and an increased incidence of mutagenic integration events.

Transgenic mice overexpressing the beta 1-adrenergic receptor in adipose tissue are resistant to obesity.

The ratio of alpha- to beta-receptors is thought to regulate the lipolytic index of adipose depots. To determine whether increasing the activity of the beta 1-adrenergic receptor (AR) in adipose tissue would affect the lipolytic rate or the development of this tissue, we used the enhancer-promoter region of the adipocyte lipid-binding protein (aP2) gene to direct expression of the human beta 1 AR cDNA to adipose tissue. Expression of the transgene was seen only in brown and white adipose tissue. Adipocytes from transgenic mice were more responsive to beta AR agonists than were adipocytes from nontransgenic mice, both in terms of cAMP production and lipolytic rates. Transgenic animals were partially resistant to diet-induced obesity. They had smaller adipose tissue depots than their nontransgenic littermates, reflecting decreased lipid accumulation in their adipocytes. In addition to increasing the lipolytic rate, overexpression of the beta 1 AR induced the abundant appearance of brown fat cells in subcutaneous white adipose tissue. These results demonstrate that the beta 1 AR is involved in both stimulation of lipolysis and the proliferation of brown fat cells in the context of the whole organism. Moreover, it appears that it is the overall beta AR activity, rather than the particular subtype, that controls these phenomena.

Mouse mammary tumor virus (MMTV), a retrovirus that exploits the immune system. Genetics of susceptibility to MMTV infection.

All animals, including humans, show differential susceptibility to infection with viruses. Study of the genetics of susceptibility or resistance to specific pathogens is most easily studied in inbred mice. We have been using mouse mammary tumor virus (MMTV), a retrovirus that causes mammary tumors in mice, to study virus/host interactions. These studies have focused on understanding the mechanisms that determine genetic susceptibility to MMTV-induced mammary tumors, the regulation of virus gene expression in vivo and how the virus is transmitted between different cell types. We have found that some endogenous MMTVs are only expressed in lymphoid tissue and that a single base pair change in the long terminal repeat of MMTV determines whether the virus is expressed in mammary gland. This expression in lymphoid cells is necessary for the infectious cycle of MMTV, and both T and B cells express and shed MMTV. Infected lymphocytes are required not only for the initial introduction of MMTV to the mammary gland, but also for virus spread at later times. Without this virus spread, mammary tumorigenesis is dramatically reduced. Mammary tumor incidence is also affected by the genetic background of the mouse and at least one gene that affects infection of both lymphocytes and mammary cells has not yet been identified. The results obtained from these studies will greatly increase our understanding of the genetic mechanisms that viruses use to infect their hosts and how genetic resistance to such viruses in the hosts occurs.

Endogenous mouse mammary tumor virus Mtv-17 is involved in Mtv-2-induced tumorigenesis in GR mice.

Mtv-2 is an endogenous mouse mammary tumor virus (MMTV) that is responsible for the induction of mammary gland tumors in the high mammary gland tumor-incidence strain GR. GR animals inherit four different endogenous MMTVs in addition to Mtv-2: Mtv-3, Mtv-7, Mtv-8 and Mtv-17. In this study we analyzed the involvement of these nonpathogenic endogenous proviruses in the mammary gland tumors caused by Mtv-2. We showed that Mtv-17 is expressed in the mammary gland of GR mice, efficiently packaged into virions, and shed into milk. DNA isolated from both mammary gland tumors and the nonmalignant mammary gland tissues of GR mice contained amplified copies of both newly acquired Mtv-2 and recombinant proviruses with the env gene derived from Mtv-17. A small percentage of these tumors contained predominantly recombinant viruses. These findings suggest a role for Mtv-17 in the mammary gland tumors induced by Mtv-2 in GR mice.