Mammary Tumor Virus Research Articles

Fibroproliferative disorders and diabetes: Understanding the pathophysiologic relationship between Peyronie's disease, Dupuytren disease and diabetes.

IntroductionFibrosis is characterized by dysregulation and accumulation of extracellular matrix. Peyronie's disease and Dupuytren disease are fibroproliferative disorders of the tunica albuginea of the penis and fascia of the hand, respectively. Chronic hyperglycaemia due to diabetes mellitus can also lead to tissue injury and fibrosis. A meta‐analysis has shown a relationship between Dupuytren disease and diabetes (overall odds ratio, 3.1; 95% confidence interval, 2.7‐3.5). This review explores commonalities in the pathogenesis of Peyronie's disease, Dupuytren disease and diabetes.MethodsA search of the PubMed database was conducted using the search terms “diabetes” AND “Peyronie's disease”; and “diabetes” AND “Dupuytren.”ResultsGenome‐wide association and gene expression studies conducted with tissue from people with Peyronie's disease or Dupuytren disease identified signalling pathways associated with wingless‐type mammary‐tumour virus integration site signalling, extracellular matrix modulation and inflammation. Biochemical studies confirmed the importance of these pathways in the pathogenesis of fibrosis with Peyronie's disease and Dupuytren disease. Dysregulation of matrix metalloproteinase activity associated with extracellular matrix breakdown was implicated in fibroproliferative complications of diabetes and in the aetiology of Peyronie's disease and Dupuytren disease. A notable percentage of people with diabetes have comorbid Peyronie's disease and/or Dupuytren disease.ConclusionsStudies have not been performed to identify fibroproliferative pathways that all 3 conditions might have in common, but data suggest that common pathways are involved in the fibroproliferative processes of Peyronie's disease, Dupuytren disease, and diabetes.

Integrin-interacting protein Kindlin-2 induces mammary tumors in transgenic mice.

Kindlin-2, an integrin-interacting protein, regulates breast cancer progression. However, currently, no animal model to study the role of Kindlin-2 in the carcinogenesis of mammary gland is available. We established a Kindlin-2 transgenic mouse model using a mammary gland-specific promoter, mammary tumor virus (MMTV) long terminal repeat (LTR). Kindlin-2 was overexpressed in the epithelial cells of the transgenic mice. The mammary gland ductal trees were found to grow faster in MMTV-Kindlin-2 transgenic mice than in control mice during puberty. Kindlin-2 promoted mammary gland growth as indicated by more numerous duct branches and larger lumens, and more alveoli were formed in the mammary glands during pregnancy under Kindlin-2 overexpression. Importantly, mammary gland-specific expression of Kindlin-2 induced tumor formation at the age of 55 weeks on average. Additionally, the levels of estrogen receptor and progesterone receptor were decreased, whereas human epidermal growth factor receptor 2 and β-catenin were upregulated in the Kindlin-2-induced mammary tumors. These findings demonstrated that Kindlin-2 induces mammary tumor formation via activation of the Wnt signaling pathway.

Assessment of androgen receptor agonistic/antagonistic effects on 25 chemicals in household applicants by OECD in vitro stably transfected transcriptional activation assays

The aim of this study is to assess the androgen receptor (AR) agonistic/antagonistic effects on various chemicals, which are used in household products including cleaning agents and wetted tissues by in vitro OECD test guideline No. 458 (using AR-EcoScreen™ cell line) and the me-too test method (using 22Rv1cell line), which was adopted as OECD project No. 4.99. All chemicals were not determined as AR agonists. However α-dodecyl-ω-hydroxypoly (oxyethylene) and 3-iodo-2-propynyl butylcarbamate have shown a weak AR antagonistic effects with IC50 values of 2.18 ± 0.12 and 4.26 ± 0.17 μg/ml via binding affinity to AR in only 22Rv1/mouse mammary tumor virus using AR transcriptional activation assay, because of their different cytotoxicity on each applied cell line. This report firstly provides information about agonistic/antagonistic effects against human AR of various chemicals including surfactants and biocides by OECD in vitro stably transfected transcriptional activation assays. However, further in vivo and human model studies are needed to confirm their adverse effects.

High prevalence of mouse mammary tumor virus-like gene sequences in breast cancer samples of Iranian women

ABSTRACTMouse mammary tumor virus (MMTV) is considered to be responsible for breast tumor development in mice. In several instances sequences homologous to the genome of this virus have been reported in human breast cancer samples.Here we aimed to evaluate MMTV involvement in human breast cancer development. DNA was extracted from 118 formalin fixed and paraffin embedded malignant (n = 59) and benign (n = 59) breast lesions. Using two sets of outer and inner MMTV-like envelope specific primers, in a nested PCR setup, MMTV genome was detected in 19 samples out of 59 (%32.2) cases of breast carcinomas, while in non-malignant breast tissue samples, only 3 samples out of 59 (%5) were positive. The difference was statistically highly significant (p < 0.001). Alignment of PCR amplified sequences with BR6, GR and C3H mouse mammary tumor virus strains emerged to be around 99% identical which is indicative of tumor tissue infection by an exogenous mouse MMTV genome.

Binding of PLD2-Generated Phosphatidic Acid to KIF5B Promotes MT1-MMP Surface Trafficking and Lung Metastasis of Mouse Breast Cancer Cells

Little is known about the cellular events promoting metastasis. We show that knockout of phospholipase D2 (PLD2), which generates the signaling lipid phosphatidic acid (PA), inhibits lung metastases in the mammary tumor virus (MMTV)-Neu transgenic mouse breast cancer model. PLD2 promotes local invasion through the regulation of the plasma membrane targeting of MT1-MMP and its associated invadopodia. A liposome pull-down screen identifies KIF5B, the heavy chain of the motor protein kinesin-1, as a new PA-binding protein. Invitro assays reveal that PA specifically and directly binds to the C terminus of KIF5B. The binding between PLD2-generated PA and KIF5B is required for the vesicular association of KIF5B, surface localization of MT1-MMP, invadopodia, and invasion in cancer cells. Taken together, these results identify a role of PLD2-generated PA in the regulation of kinesin-1 motor functions and breast cancer metastasis and suggest PLD2 as a potential therapeutic target for metastatic breast cancer.

Single amino acid substitution (G42E) in the receptor binding domain of mouse mammary tumour virus envelope protein facilitates infection of non-murine cells in a transferrin receptor 1-independent manner.

BackgroundMouse mammary tumour virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse tranferrin receptor 1 (TfR1) for cell entry. Several MMTV strains have been shown to productively infect, in addition to murine cells, various heterologous cell lines including those of human origin, albeit less efficiently than murine cells. Furthermore, there have been reports that the continued passage of MMTV in heterologous cell lines gives rise to novel variants that are able to infect naive non-murine cells with higher efficiency than the parental virus.ResultsWe show that MMTV(C3H), like other MMTV strains, that had undergone a number of replication cycles in non-murine cells displayed an increased replication kinetic, as compared to parental virus, when applied on naive human cells. Sequence analysis of several replication kinetic variants and the parental virus, together with calculation of the ratio of non-synonymous to synonymous mutations at individual codons, revealed that several regions within the viral genome were under strong positive selection pressure during viral replication in human cells. The mutation responsible, at least in part, for the phenotypic change was subsequently mapped to the segment of env encoding the receptor binding site (F40HGFR44). Introduction of the identified mutation, leading to single amino acid substitution (G42E), into egfp-containing recombinant MMTV virions enhanced their ability to bind to and infect human cells. Interestingly, neither the replication kinetic mutant nor the parental virus required human TfR1 for infection. Knock-out of TFR1 gene from the human genome did not decrease the susceptibility of Hs578T cells to virus infection. Furthermore, the expression of human TfR1, in contrast to mouse TfR1, did not enhance the susceptibility of MMTV-resistant Chinese hamster ovary cells. Thus, human TfR1 is dispensable for infection and another cell surface molecule mediates the MMTV entry into human cells.ConclusionTaken together, our data explain the mechanism enabling MMTV to form ‘host-range variants’ in non-murine cells that has been known for a long time, the basis of which remained obscure. Our findings may expand our understanding of how viruses gain capability to cross species-specific barriers to infect new hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0168-2) contains supplementary material, which is available to authorized users.

Abstract 530: MicroRNA expression profiling in MMTV-neu breast cancer mouse model

Abstract In addition to mRNA gene expression profiling, miRNA expression analyses of human breast cancers have further demonstrated another layer of the molecular diversity of this disease and may potentially be a useful diagnostic and prognostic tool for breast cancer therapy and treatment. The development and use of relevant mouse models that closely reflect genetic alterations found in human breast cancers is critical to understand malignant progression in this disease and to test developmental therapeutics. The Mammary Tumour Virus (MMTV) Long Terminal Repeat (LTR) promoter-driven HER2/neu transgenic mouse model was developed to directly assess the effects of mammary gland specific expression of the neu protooncogene. Global miRNA expression profiling was performed on tumours derived from the MMTV-HER2/neu model. Normal mammary glands from FVB wild-type mice, HER2 overexpressing mammary glands from MMTV-HER2/neu transgenic mice and tumours derived from this model at different stages of the tumourigenic process were collected and microRNA expression profiling performed on them by Nanostring analysis. Differential expression of selected miRNAs, obtained by Nanostring screening, was further confirmed by quantitative Real Time analysis. By analyzing global miRNA expression profile on tissues collected from MMTV-HER2/neu mice at different stages of the tumourigenesis, we identified significant differences in miRNA expression patterns between tumours and normal mammary tissue and between non-metastatic and metastatic tumours. miRNA expression characterization of tumours at various stages of development along with gene expression in mouse models would profoundly enhance our knowledge of progressive genetic changes associated with oncogene-induced events in breast cancer. Citation Format: Claudia Piovan, Gianpiero Di Leva, Douglas Cheung, Matteo Fassan, Arpan Kumar, Dario Palmieri, Marilena V. Iorio, Carlo M. Croce. MicroRNA expression profiling in MMTV-neu breast cancer mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 530. doi:10.1158/1538-7445.AM2014-530

119. Psychological stressors significantly speed the development of primary mammary tumors in the HER-2/neu mouse transgenic breast cancer model

The transgenic mouse strain FVB/N-Tg (MMTVneu)202Mul/J has been characterized as the “workhorse model of HER2 tumorigenesis in vivo.” These mice selectively express rat c-Neu/ErbB2 (HER2-neu) in their mammary glands via a mammary tumor virus (MMTV) promoter, which is responsive in cell cuture to glucocorticoids. We tested the hypothesis that exposure to environmental stressors in vivo would result in early onset of mammary tumors in this strain. Adult female mice, 4–5 weeks of age, were purchased; housed in an isolation chamber, and assigned to four experimental exposure groups: Restraint stress (90 min, 1x/wk, n = 24)[RS]; Social Disruption (cage mate switch 2x/wk, n = 23)[SD]; Restraint + Social Disruption (n = 22) and Home Cage controls (n = 20)[HC]. All mice were inspected (blind) for tumors weekly and humanely sacrificed one week post palpable tumor development. Consistent with the study hypothesis, the results indicated that mice chronically exposed to the two environmental stressors (RS& SD) developed primary tumors at a significantly (p

Abstract A109: A signal peptide as a putative tool against MMTV-associated breast cancers

Abstract Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma and lymphoma in mice. An increasing body of evidence from different laboratories suggests an association between MMTV and up to 38% of human breast cancers. This seems related to severity of the disease and geographical location. Signal peptides are N-terminal extensions of nascent secretory and membrane proteins (typically including 15-25 amino acid residues). They mediate translocation across, or insertion into the membrane of the endoplasmic reticulum (ER). The signal peptide of Mouse Mammary Tumor Virus (MMTV) envelop precursor (named MMTV-14 or p14 for short), in addition to being unusually long (98 a.a) demonstrates some unique characteristics: 1) It localizes to nucleoli; 2) It functions as a nuclear export factor for intron-containing viral transcripts; 3) It can function in a pro-oncogenic or anti-oncogenic capacity depending on its phosphorylation by the kinases CK2 (at serine 65) or PKC (at serine 18), respectively. We report that, in addition to its nucleolar localization, p14 is expressed on the cell surface of murine lymphomas and mammary carcinomas that harbor MMTV, as well as on the surface of MCF-7 human breast cancer cells ectopically expressing p14. Cell surface expression allows p14 to play the role of a tumor associated antigen. Indeed, priming and boosting of mice with recombinant p14, using different adjuvants, induces specific anti-p14 antibodies, as well as immunizes these mice against malignant tumor cells that harbor MMTV. This immunity is adoptively transferred to naïve mice. Thus, MMTV-p14 is a multi-faceted signal peptide with immune modulating characteristics that can be used for developing both preventive and therapeutic approaches towards MMTV-associated breast cancers. Supported in part by a Kamin grant from the Office of the Chief Scientist. Citation Format: Ori Braitbard, Allan Bar-Sinai, Maayan Roniger, Dana Rajchman, Nira Hochman, Jacob Hochman. A signal peptide as a putative tool against MMTV-associated breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A109.

The Signal Peptide of Mouse Mammary Tumor Virus-Env: A Phosphoprotein Tumor Modulator

Mouse mammary tumor virus (MMTV) is associated primarily with mammary carcinomas and lymphomas. The signal peptide of the MMTV envelope precursor is uniquely targeted to nucleoli of cells that harbor the virus, where it can function as a nuclear export factor for intron-containing transcripts. Antibodies to this signal peptide, which we refer to as p14, were previously shown to label nucleoli in a subset of human breast cancers. To look for additional cellular functions of p14, different mutants were ectopically expressed in the MCF-7 human breast cancer cell line. This approach identified motifs responsible for its nucleolar targeting, nucleocytoplasmic shuttling, target protein (B23, nucleophosmin) binding, and phosphorylation at serine 18 and 65 both in situ and in vitro. To test the role of these phosphorylation sites, we carried out in vivo tumorigenesis studies in severe combined immunodeficient mice. The findings show that the p14-Ser65Ala mutation is associated with impaired tumorigenicity, whereas the p14-Ser18Ala mutation is associated with enhanced tumorigenicity. Microarray analysis suggests that phosphorylation at serine 18 or at serine 65 is associated with transcriptional regulation of the L5 nucleolar ribosomal protein (a p14 target) and the Erb-B signal transduction pathway. Taken together, these results show that the phosphorylation status of p14 determines whether it functions as a pro-oncogenic or antioncogenic modulator.

Requirements for Mouse Mammary Tumor Virus Rem Signal Peptide Processing and Function

Mouse mammary tumor virus (MMTV) encodes a Rev-like protein, Rem, which is involved in the nuclear export and expression of viral RNA. Previous data have shown that all Rev-like functions are localized to the 98-amino-acid signal peptide (SP) at the N terminus of MMTV Rem or envelope proteins. MMTV-SP uses endoplasmic reticulum-associated degradation (ERAD) for protein trafficking. Rem cleavage by signal peptidase in the ER is necessary for MMTV-SP function in a reporter assay, but many requirements for trafficking are not known. To allow detection and localization of both MMTV-SP and the C-terminal cleavage product, we prepared plasmids expressing green fluorescent protein (GFP) tags. N-terminal Rem tagging led to protein accumulation relative to untagged Rem and allowed signal peptidase cleavage but reduced its specific activity. C-terminal tagging also led to Rem accumulation yet dramatically reduced cleavage, GFP fluorescence, and activity relative to N-terminally tagged Rem (GFPRem). Substitutions of an invariant leucine at position 71 between the known RNA-binding and nuclear export sequences interfered with GFPRem accumulation and activity but not cleavage. Similarly, deletion of 100 or 150 C-terminal amino acids from GFPRem dramatically reduced both Rem and MMTV-SP levels and function. Removal of the entire C terminus (203 amino acids) restored both protein levels and activity of MMTV-SP. Only C-terminal GFP tagging, and not other modifications, appeared to trap Rem in the ER membrane. Thus, Rem conformation in both the ER lumen and cytoplasm determines cleavage, retrotranslocation, and MMTV-SP function. These mutants further characterize intermediates in Rem trafficking and have implications for all proteins affected by ERAD.

Mouse Mammary Tumor Virus-Like Nucleotide Sequences in Canine and Feline Mammary Tumors

Mouse mammary tumor virus (MMTV) has been speculated to be involved in human breast cancer. Companion animals, dogs, and cats with intimate human contacts may contribute to the transmission of MMTV between mouse and human. The aim of this study was to detect MMTV-like nucleotide sequences in canine and feline mammary tumors by nested PCR. Results showed that the presence of MMTV-like env and LTR sequences in canine malignant mammary tumors was 3.49% (3/86) and 18.60% (16/86), respectively. For feline malignant mammary tumors, the presence of both env and LTR sequences was found to be 22.22% (2/9). Nevertheless, the MMTV-like LTR and env sequences also were detected in normal mammary glands of dogs and cats. In comparisons of the MMTV-like DNA sequences of our findings to those of NIH 3T3 (MMTV-positive murine cell line) and human breast cancer cells, the sequence similarities ranged from 94 to 98%. Phylogenetic analysis revealed that intermixing among sequences identified from tissues of different hosts, i.e., mouse, dog, cat, and human, indicated the MMTV-like DNA existing in these hosts. Moreover, the env transcript was detected in 1 of the 19 MMTV-positive samples by reverse transcription-PCR. Taken together, our study provides evidence for the existence and expression of MMTV-like sequences in neoplastic and normal mammary glands of dogs and cats.

Mouse Mammary Tumor Virus–like Sequences in Human Breast Cancer

Mouse mammary tumor virus (MMTV) sequences have been reported to be present in some human breast cancers, but it is unclear whether they have any causal role. In mice, MMTV promotes tumor formation indirectly by insertional mutagenesis of Wnt oncogenes that lead to their activation. In this study, we investigated the status of Wnt-1 in human breast cancers harboring MMTV-like sequences encoding viral envelope (env) genes. We confirmed the detection of env sequences in the nucleus of human breast cancer specimens that are similar in appearance to mouse mammary tumors expressing MMTV env sequences. MMTV env sequences in human breast cancers were also nearly indistinguishable from env sequences in mouse MMTV isolates. Further, Wnt-1 expression was higher in specimens of env-positive ductal carcinoma in situ and invasive ductal carcinoma, relative to env-negative specimens. Our findings extend the evidence that MMTV sequences found in naturally occurring mouse mammary tumors can be found in some human breast cancers, prompting further evaluation of causal roles in these settings.

Rev and Rex proteins of human complex retroviruses function with the MMTV Rem-responsive element

BackgroundMouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE).ResultsMMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export.ConclusionThese data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.

The history of tumor virology.

In the century since its inception, the field of tumor virology has provided groundbreaking insights into the causes of human cancer. Peyton Rous founded this scientific field in 1911 by discovering an avian virus that induced tumors in chickens; however, it took 40 years for the scientific community to comprehend the effect of this seminal finding. Later identification of mammalian tumor viruses in the 1930s by Richard Shope and John Bittner, and in the 1950s by Ludwik Gross, sparked the first intense interest in tumor virology by suggesting the possibility of a similar causal role for viruses in human cancers. This change in attitude opened the door in the 1960s and 1970s for the discovery of the first human tumor viruses--EBV, hepatitis B virus, and the papillomaviruses. Such knowledge proved instrumental to the development of the first cancer vaccines against cancers having an infectious etiology. Tumor virologists additionally recognized that viruses could serve as powerful discovery tools, leading to revolutionary breakthroughs in the 1970s and 1980s that included the concept of the oncogene, the identification of the p53 tumor suppressor, and the function of the retinoblastoma tumor suppressor. The subsequent availability of more advanced molecular technologies paved the way in the 1980s and 1990s for the identification of additional human tumor viruses--human T-cell leukemia virus type 1, hepatitis C virus, and Kaposi's sarcoma virus. In fact, current estimates suggest that viruses are involved in 15% to 20% of human cancers worldwide. Thus, viruses not only have been shown to represent etiologic agents for many human cancers but have also served as tools to reveal mechanisms that are involved in all human malignancies. This rich history promises that tumor virology will continue to contribute to our understanding of cancer and to the development of new therapeutic and preventive measures for this disease in the 21st century.

Early Increases in Superantigen-Specific Foxp3+Regulatory T Cells during Mouse Mammary Tumor Virus Infection

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Here, we show in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (SAg)-specific Foxp3(+) regulatory T cells (T(reg)) in Peyer's patches (PP). These increases were shown to be dependent on the presence of dendritic cells. CD4(+) CD25(+) T cells from the PP of infected mice preferentially suppress the proliferative response of T cells to SAg-expressing antigen-presenting cells ex vivo. We investigated the influence of the depletion of CD25(+) cells at different stages of the infection. When CD25(+) cells were depleted before MMTV infection, an increase in the number of PP SAg-cognate Foxp3(-) T cells was found at day 6 of infection. Since the SAg response is associated with viral amplification, the possibility exists that T(reg) cells attenuate the increase in viral load at the beginning of the infection. In contrast, depletion of CD25(+) cells once the initial SAg response has developed caused a lower viral load, suggesting that at later stages T(reg) cells may favor viral persistence. Thus, our results indicated that T(reg) cells play an important and complex role during MMTV infection.

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.

In Utero Transplantation of Transduced Hematopoietic Stem Cells Results in Deletion of Transgene-Specific T Cells.

The developing fetal immune system provides a unique opportunity to manipulate normal immunologic development for therapeutic prenatal and anticipated postnatal interventions. In previous studies we have shown that allogeneic in utero hematopoietic cell transplantation (IUHCT) results in donor specific tolerance that can subsequently facilitate non-myeloablative postnatal cellular or organ transplants. It follows that in utero injection of transduced hematopoietic stem cells (HSC) could potentially induce tolerance to a transgene encoded protein. We hypothesized that expression of a transduced antigenic protein by HSC and their progeny would alter thymic T cell development resulting in deletion of antigen specific T-cells. To test this hypothesis, we used the mammary tumor virus (MTV) superantigen system to evaluate the effect of IUHCT of transduced HSC on T cell development. In this system, expression of different MTV oncogenes by different I-E+ strains of mice results in deletion of T cells expressing the relevant Vβ T cell receptor. Specifically, mice which are Mtv7+ delete T cells expressing the Vβ6 T-cell receptor. In this study, CD150+CD48− enriched Balb/c (I-E+ Mtv7−) HSC were transduced with an HIV-based lentivirus expressing MTV7 under an MND promoter. 1.5E+05 transduced cells were injected intravascularly via the vitelline vein into E14 Balb/c fetuses. Non-injected age matched naive Balb/c mice served as the control group. The peripheral blood (PB) and thymuses of injected fetuses and control mice were harvested at day of life (DOL) 10, 20 and 60 and analyzed by flow cytometry for T lymphocyte Vβ6 expression. Additionally, the T cell composition of the thymus was assessed at DOL10 for CD4 and CD8 single positive (SP) and CD4/CD8 double positive (DP) cells. Thymic flow cytometric analysis at DOL10 revealed that greater than 98% of the T cells were CD4CD8 DP, a stage that has not yet undergone negative selection. No significant difference was noted in the percentage of thymic Vβ6+ DP T-cells at this time point or at DOL20 and DOL60. In contrast, there was a significant decrease in the percentage of Vβ6+ peripheral blood SP cells in those mice injected with MTV7 transduced HSC relative to control mice at DOL10, DOL20 and DOL60 (p<0.05) (Fig 1). The current study supports the ability of enriched transduced HSC to induce deletion of transgene specific T cells after IUHCT. In the future, this strategy may be useful to promote tolerance for pre or postnatal cellular or gene therapy. [Display omitted]

An Immunoreceptor Tyrosine Activation Motif in the Mouse Mammary Tumor Virus Envelope Protein Plays a Role in Virus-Induced Mammary Tumors

Mouse mammary tumor virus (MMTV) induces breast cancer with almost 100% efficiency in susceptible strains through insertional activation of protooncogenes, such as members of the wnt and fibroblast growth factor (fgf) families. We previously showed that expression of the MMTV envelope protein (Env) in normal immortalized mammary epithelial cells grown in three-dimensional cultures caused their morphological transformation, and that this phenotype depended on an immunoreceptor tyrosine-based activation motif (ITAM) present in Env and signaling through the Syk tyrosine kinase (E. Katz, M. H. Lareef, J. C. Rassa, S. M. Grande, L. B. King, J. Russo, S. R. Ross, and J. G. Monroe, J. Exp. Med. 201:431-439, 2005). Here, we examined the role of the Env protein in virus-induced mammary tumorigenesis in vivo. Similar to the effect seen in vitro, Env expression in the mammary glands of transgenic mice bearing either full-length wild-type provirus or only Env transgenes showed increased lobuloalveolar budding. Introduction of the ITAM mutation into the env of an infectious, replication-competent MMTV or into MMTV/murine leukemia virus pseudotypes had no effect on incorporation of Env into virus particles or on in vitro infectivity. Moreover, replication-competent MMTV bearing the ITAM mutation in Env infected lymphoid and mammary tissue at the same level as wild-type MMTV and was transmitted through milk. However, mammary tumor induction was greatly attenuated, and the pattern of oncogene activation was altered. Taken together, these studies indicate that the MMTV Env protein participates in mammary epithelial cell transformation in vivo and that this requires a functional ITAM in the envelope protein.

Challenge with mammary tumor cells expressing MHC class II and CD80 prevents the development of spontaneously arising tumors in MMTV-neu transgenic mice

The HER-2/Neu oncogene has been implicated in human and mouse breast cancer. Indeed, transgenic MMTV-neu mice expressing this oncogene from the mammary tumor virus long terminal repeat develop spontaneous mammary tumors and die within 1 year of life. We have expressed the class II transactivator (CIITA) and/or the costimulatory molecule CD80 (B7.1) in a mammary carcinoma cell line (MCNeuA) derived from these mice. Class II transactivator directs the expression of MHC class II and the machinery for antigen processing and presentation by this pathway. When injected into MMTV-neu mice, tumor cells expressing CD80 or CD80 and CIITA, were rejected completely. In addition, following the rejection of dual expressing cells, 75% of the mice were protected against the development of subsequent spontaneous tumors. Cells expressing only CD80 or CIITA were not as effective as antitumor vaccines in preventing the development of spontaneous tumors. Thus, converting cancer cells into antigen presenting cells could represent an effective immunotherapy for breast cancer.