Localization of peripheral benzodiazepine binding sites and diazepam-binding inhibitor (DBI) mRNA in mammary glands and dimethylbenz(a)antracene (DMBA)-induced mammary tumors in the rat.

An association of diazepam-binding inhibitor (DBI), an endogenous ligand at the benzodiazepine (BZD) receptor, with the peripheral type BDZ receptor (PBR) has been reported in the brain and a few peripheral tissues. In order to verify whether or not DBI and PBR are present in the mammary tissue, we have proceeded to the localization of DBI mRNA and PBR in rat mammary glands and DMBA-induced mammary tumors. DBI mRNA was detected by in situ hybridization using a 35S-labelled single-stranded RNA probe complementary to DBI mRNA and PBR by in vitro autoradiography using [3H]PK11195 as the ligand. In mammary glands from virgin and lactating animals, both DBI mRNA and PBR were detected in acinar cells. In dimethylbenz(a)anthracene (DMBA)-induced tumors, hybridization signal was not detected in all the cells whereas PBR appeared to be present in all the tumoral cells, although non uniformly distributed. These data indicating that mammary DMBA-induced tumoral cells contain both DBI and PBR suggest that BZD receptors might be involved in the regulation of mammary glands as well as mammary tumoral cells.     

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.     

Mouse mammary tumor virus-induced tumorigenesis in sag transgenic mice: a laboratory model of natural selection.

Transgenic mice that expressed the superantigen protein encoded in the C3H exogenous mouse mammary tumor virus long terminal repeat deleted their V beta 14+ T cells during the shaping of their immune repertoire and showed no evidence of virus production in their mammary glands after infection by milk-borne C3H exogenous virus. However, they developed mammary gland tumors that had newly integrated copies of C3H exogenous virus, although the latency of tumor formation was much longer than in their nontransgenic littermates that retained their V beta 14+ T cells. After four generations, infectious C3H virus was completely eliminated from the transgenic mouse pedigree. These data support the hypothesis that endogenous mouse mammary tumor proviruses are retained in the genome as protection against exogenous virus infection and subsequent tumorigenesis and show that there may be natural selection against the virus in vivo.     

Nonuniform expression of a mouse mammary tumor virus-driven int-2/Fgf-3 transgene in pregnancy-responsive breast tumors.

We have developed transgenic mice in which expression of the mouse int-2/Fgf-3 gene is regulated by a single long terminal repeat from mouse mammary tumor virus. Such mice contain and transmit a replica of the activated int-2/Fgf-3 allele present in a spontaneous mammary tumor from a BR6 mouse. Although free of infectious mouse mammary tumor virus and with a different genetic background, the transgenic mice develop pregnancy-responsive mammary epithelial proliferations that are similar to the early stages of tumorigenesis in the BR6 strain. Histological examination revealed that most of these tumors showed pronounced tubular and acinar structures, features usually associated with morphological differentiation. In some cases, the tumors were locally invasive, causing disruption of the dermis which manifested itself as local hair loss. In situ hybridization showed that patterns of transgene expression in the abnormal glands were markedly nonuniform. In contrast, mouse mammary tumor virus-induced neoplasms showed more uniform expression of int-2/Fgf-3, as did the urogenital epithelial proliferations that occur among males of this transgenic line. These data suggest that mammary tumors in virally infected animals may depend primarily on autocrine stimulation by the int-2/Fgf-3 gene product, whereas both autocrine and paracrine mechanisms may contribute to tumors and hyperplasias found in transgenic animals.     

Stochastic appearance of mammary tumors in transgenic mice carrying the MMTV/c-neu oncogene

Transgenic mice carrying the activated c-neu oncogene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat were produced. Epithelial hyperplasia of epididymis, seminal vesicles, and salivary glands, and dysplasia of harderian glands, were induced. Moreover, in females of our four lines, independent but multiple mammary tumors arose asynchronously, between 5 and 10 months of age, as stochastic events. Histologically, poorly differentiated adenocarcinomas, with intratumor necrosis and calcifications, arose adjacent to morphologically normal epithelium. High transgene expression was detected in all mammary tumors tested and in normal mammary glands before the appearance of the tumors. Together these results suggest that the expression of the activated c-neu oncogene was necessary but not sufficient to induce malignant transformation of the mammary epithelial cells. These tumors appear to be an adequate model for human breast cancers overexpressing c-neu.     

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).     

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.     

Human adenovirus type 9-induced rat mammary tumors.

Following subcutaneous inoculation of newborn Wistar-Furth rats with human adenovirus type 9 (Ad9), 16 of 16 female and 0 of 11 male rats developed mammary tumors. Tumor-positive animals usually developed tumors in multiple glands. Histopathological analyses indicated that three general categories of tumor could be identified. Mammary fibroadenomas were the most common tumor type encountered, but phyllodeslike tumors and solid sarcomas were also frequently found. In situ hybridization and immunohistochemical techniques established that benign fibroadenomas were derived from mammary fibroblasts (collagen type I- and vimentin-positive cells) and that malignant tumors were derived from myoepithelial cells (collagen type IV-, vimentin-, and muscle-specific actin-positive cells). The fact that mammary tumors were limited to female rats suggested that female hormones are essential for tumor growth and development. In this regard, ovariectomy of Ad9-infected female rats prevented tumor development, while subsequent diethylstilbestrol (DES) treatment elicited tumor formation. In addition, Ad9-infected and castrated male rats which received DES also developed mammary tumors. Established male mammary tumors regressed when DES treatment was stopped and reappeared after DES treatment was resumed. Together, these results indicate that estrogen is required for both initiation and maintenance of Ad9-induced mammary tumors. Southern blot analysis of high-molecular-weight tumor DNA showed that mammary tumor cells contained single or multiple integrated copies of the entire Ad9 genome. RNase protection experiments established that estrogen receptor as well as Ad9 E1a and E4 mRNAs were expressed in mammary tumors, but Ad9 E3 and, surprisingly, E1b mRNAs were not expressed at detectable levels.     

Relationship Between Organization of Mammary Tumors and the Ability of Tumor Cells to Replicate Mammary Tumor Virus and to Recognize Growth-Inhibitory Contact Signals In Vitro

Mammary tumor virus (MTV) replication was confined primarily to cells organized as acini in intact mouse mammary glands. Primary mammary tumors maintained a high degree of acinar organization and cells therein continued to replicate MTV vegetatively. Nonacinar mammary cells, derived by serial transplantation of acinar tumor cells, no longer actively replicated MTV. This suggests that phenotypic differences exist among mammary epithelial cells in their ability to support virus replication, that a fundamental relationship exists between the organization of epithelium for secretion and active virus replication, and that this relationship is not altered as a primary consequence of neoplastic transformation. Mammary epithelial cells from pregnant, non-tumor-bearing, MTV-infected BALB/cfC3H mice or from acinar mammary tumors from a number of mouse strains were grown in primary monolayer cultures. Such cell cultures under the influence of insulin and cortisol exhibited the ability to organize into discrete three-dimensional structures called "domes." MTV replication in such cultures took place primarily in cells within the organized domes. Cells cultured from nonacinar tumors did not exhibit any propensity to organize into domes, nor did they replicate MTV in primary culture. This suggests that the cell organizational requirement for MTV replication observed in vivo is conserved in primary culture. Dome formation is not an effect of virus replication, as cells from uninfected BALB/c animals organized into domes in culture without concomitant MTV replication. Growth-regulating signals, exerted between contiguous cells in cultures of non-MTV-infected mammary epithelium, were not modified by the occurrence of active virus replication nor as a direct consequence of neoplastic transformation. Cells derived from nontumor BALB/cfC3H glands and from spontaneous tumors exhibited cell growth kinetics, saturation densities, and deoxyribonucleic acid synthesis kinetics nearly identical to those of noninfected normal mammary epithelium in primary culture. Cell to cell growth regulatory signals were modified in cultures of nonalveolar tumor cells wherein evidence of overgrowth is documented.     

Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis

Emerging data suggest that metastasis-associated protein 1 (MTA1) represses ligand-dependent transactivation functions of estrogen receptor-alpha in cultured breast cancer cells and that MTA1 is upregulated in human breast tumors. However, the role of MTA1 in tumorigenesis in a physiologically relevant animal system remains unknown. To reveal the role of MTA1 in mammary gland development, transgenic mice expressing MTA1 under the control of the mouse mammary tumor virus promoter long terminal repeat were generated. Unexpectedly, we found that mammary glands of these virgin transgenic mice exhibited extensive side branching and precocious differentiation because of increased proliferation of ductal and alveolar epithelial cells. Mammary glands of virgin transgenic mice resemble those from wild-type mice in mid-pregnancy and inappropriately express beta-casein, cyclin D1 and beta-catenin protein. Increased ductal growth was also observed in the glands of ovariectomized female mice, as well as of transgenic male mice. MTA1 dysregulation in mammary epithelium and cancer cells triggered downregulation of the progesterone receptor-B isoform and upregulation of the progesterone receptor-A isoform, resulting in an imbalance in the native ratio of progesterone receptor A and B isoforms. MTA1 transgene also increased the expression of progesterone receptor-A target genes Bcl-XL (Bcl2l1) and cyclin D1 in mammary gland of virgin mice, and, subsequently, produced a delayed involution. Remarkably, 30% of MTA1 transgenic females developed focal hyperplastic nodules, and about 7% exhibited mammary tumors within 18 months. These studies establish, for the first time, a potential role of MTA1 in mammary gland development and tumorigenesis. The underlying mechanism involves the upregulation of progesterone receptor A and its targets, Bcl-XL and cyclin D1.     

Involvement of mouse mammary tumor virus in spontaneous and hormone-induced mammary tumors in low-mammary-tumor mouse strains.

The involvement of the mouse mammary tumor virus (MTV) in spontaneous and hormone-induced mammary tumors in low-mammary-tumor mouse strains was studied by comparing the amounts of MTV RNA and MTV DNA sequences in mammary tumors and other tissues of mice with an without hormonal treatments. The following results were obtained. (i) Mammary tumors which appeared in C3H mice as a result of an infection with MTV contained more MTV DNA compared with noninfected organs; these mammary tumors also contained more MTV RNA than was present in lactating mammary gland cells. (ii) Hormonal stimulation by administration of excessive amounts of prolactin via hypophyseal isografts in C3Hf and O20 mice resulted in an increased expression of MTV RNA in the mammary glands. This elevated level of MTV RNA expression was, however, not maintained in the hormone-induced mammary tumors. (iii) Spontaneous mammary tumors in BALB/c mice contained similar levels of MTV DNA and MTV RNA sequences as were found in other cells of these animals.     

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.     

A replication-competent promoter-trap retrovirus.

A promoter-trap retrovirus has been constructed in which a promoterless polyomavirus middle T antigen gene was inserted in the U3 region of the long terminal repeat of a replication-competent Moloney murine leukemia virus. The resulting virus, designated PyT, was used to infect mouse mammary glands in situ. As expected, mammary tumors appeared in some infected animals. These tumors were found to contain PyT proviruses of the predicted structure. From one such tumor, the PyT provirus and surrounding sequences from the integration site were cloned. The provirus was found to have integrated adjacent to the promoter of a novel mouse gene (TRAP1) that was expressed at low levels in various mouse tissues. These data show that the PyT retrovirus provides a sensitive means of detecting active promoters in vivo.     

Murine Mammary Tumor Virus Expression During Mammary Tumorigenesis in BALB/c Mice

Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.     

Telomerase activation in mouse mammary tumors: lack of detectable telomere shortening and evidence for regulation of telomerase RNA with cell proliferation.

Activation of telomerase in human cancers is thought to be necessary to overcome the progressive loss of telomeric DNA that accompanies proliferation of normal somatic cells. According to this model, telomerase provides a growth advantage to cells in which extensive terminal sequence loss threatens viability. To test these ideas, we have examined telomere dynamics and telomerase activation during mammary tumorigenesis in mice carrying a mouse mammary tumor virus long terminal repeat-driven Wnt-1 transgene. We also analyzed Wnt-1-induced mammary tumors in mice lacking p53 function. Normal mammary glands, hyperplastic mammary glands, and mammary carcinomas all had the long telomeres (20 to 50 kb) typical of Mus musculus and did not show telomere shortening during tumor development. Nevertheless, telomerase activity and the RNA component of the enzyme were consistently upregulated in Wnt-1-induced mammary tumors compared with normal and hyperplastic tissues. The upregulation of telomerase activity and RNA also occurred during tumorigenesis in p53-deficient mice. The expression of telomerase RNA correlated strongly with histone H4 mRNA in all normal tissues and tumors, indicating that the RNA component of telomerase is regulated with cell proliferation. Telomerase activity in the tumors was elevated to a greater extent than telomerase RNA, implying that the enzymatic activity of telomerase is regulated at additional levels. Our data suggest that the mechanism of telomerase activation in mouse mammary tumors is not linked to global loss of telomere function but involves multiple regulatory events including upregulation of telomerase RNA in proliferating cells.     

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.     

The role of humoral and cellular mediators in enhanced mammary inflammatory reactions to staphylococcal infection in systemically immunized ewes

Prior systemic immunization with live Staphylococcus aureus vaccine enhances the early recruitment of neutrophils into nonlactating mammary glands infected with staphylococci. The study investigates the role of humoral and cellular mediators in this phenomenon. Intramammary infusion of bacteria suspended in immune sheep serum did not enhance the inflammatory response to infection in nonimmunized ewes despite the presence of complement in the infused serum. Infusion of complement activated by incubation with zymosan evoked a massive neutrophil influx into mammary secretions by 4 hr after infusion. Hemolytic complement activity was not detected in mammary secretions of immunized or nonimmunized ewes. These findings indicate that, despite the inflammatory effect of complement activation, humoral immune factors did not promote neutrophil migration into infected glands. Mammary glands of systemically immunized ewes stimulated 5 days previously with staphylococcal soluble antigens (SSA) supported larger neutrophil influxes during staphylococcal infection than contralateral glands stimulated with endotoxin 5 days prior to infection. Exudates of SSA-stimulated glands had significantly higher cell concentrations, prior to infection, than endotoxin-stimulated glands; however elevated cell concentrations in endotoxin-stimulated glands of nonimmune ewes did not support enhanced inflammatory responses. These findings suggest that qualitative but not quantitative characteristics of mammary leucocytes influence the inflammatory response to infection in systemically immunized ewes.     

Endogenous MMTV in the normal mammary gland and in dimethylanthracene-induced mammary cancer in BALB/c mice

Immunization of DMBA-treated mice by the glycoprotein fraction from mammary glands of mice BALB/c did not decrease the frequencies of induction of mammary tumors. This is in contrast to the results obtained during immunization by the formaldehyde treated preparation of Mouse Mammary Tumor Virus (MMTV). EcoRI and HindIII cleaved DNA from the DMBA-induced mammary tumors did not contain the additional virus specific fragments. In mammary tumors the expression of p27 MMTV was registered in contrast to normal mammary glands and mammary epithelium cultures in which the proteins of MMTV are not expressed even after induction.