Endothelial cell tumors develop in transgenic mice carrying polyoma virus middle T oncogene

Inoculation of newborn mice with the murine polyoma (Py) virus leads to tumor formation in a wide range of tissues. In order to investigate viral oncogenesis, we generated transgenic mice carrying either the Py large T antigen (LT) gene or the Py middle T antigen (MT) gene linked to Py early region regulatory sequences. While Py LT mice exhibit no phenotype, Py MT mice develop multifocal tumors of the vascular endothelium. These hemangiomas are lethal to the animals and can be passaged in vivo. Transgene RNAs and protein are present in both hemangiomas and the testes of these mice, and the Py middle T protein in both tissues is complexed to a cellular tyrosine kinase. The expression of complexed middle T protein in both tumorigenic endothelial cells and unperturbed testes implies that endothelial cells may be particularly susceptible to the action of the middle T oncogene. These observations indicate that Py middle T disrupts the normal strict controls on vascular growth, and suggest that Py MT transgenic mice will provide a model for studying the control of angiogenesis.     

Ret transgenic mouse model of spontaneous skin melanoma: focus on regulatory T cells

Ret transgenic mouse model of skin malignant melanoma is characterized by the overexpression of the human ret transgene in melanin‐containing cells. Transgenic mice spontaneously develop skin tumors with metastases in lymph nodes, lungs, liver, brain, and the bone marrow. Tumor lesions show typical melanoma morphology and express melanoma‐associated antigens. Although transgenic mice demonstrate an accumulation of melanoma antigen‐specific memory and effector T cells, their anti‐tumor effects could be blocked by highly immunosuppressive leukocytes enriched in the tumor microenvironment and in the periphery. Here, we discuss the role of one of the most potent immunosuppressive subset, regulatory T cells, in the melanoma progression in this model.     

Differentiation and oncogenesis: phenotypically distinct lens tumors in transgenic mice

We report on two lines of transgenic mice that express a murine alpha A-crystallin/SV40 tumor antigen fusion gene in the eye lens. The alpha T1 line develops fast growing, poorly differentiated lens tumors, whereas the alpha T2 line produces lens tumors that are slow growing and well differentiated. There is a striking difference between these two lines in the temporal and spatial patterns of tumor antigen expression during initial lens development. In the alpha T1 line, the transgene is expressed very early in development in most lens cells, and no primary fiber differentiation takes place. In the alpha T2 line, transgene expression occurs after primary fiber formation has been initiated, and is restricted to differentiating fiber cells. The anterior epithelium from both alpha T lines undergoes normal development and remains morphologically normal until after birth, although in alpha T1 mice, these anterior cells produce considerable amounts of SV40 tumor antigens. This suggests that the state of differentiation of the lens cell plays an important role in its response to oncogene products.     

Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo

In order to track hematopoetic cells of all lineages unambiguously at all stages of development, we have developed C57BL/6 mice that express a transgene coding for green fluorescent protein (GFP) under control of the human ubiquitin C promoter. These mice, called UBI-GFP/BL6, express GFP in all tissues examined, with high levels of GFP expression observed in hematopoetic cells. UBI-GFP/BL6 mice are unique in that B cells, T cells, and dendritic cells have distinct levels of GFP fluorescence. In cell transfer experiments, leukocytes from UBI-GFP/BL6 mice are readily identified by FACS or fluorescence microscopy. We demonstrate that transplanted UBI-GFP/BL6 dendritic cells are easily identified in secondary lymphoid tissues. Direct interactions between individual dendritic cells and multiple na?ve CD8+ T cells are observed in lymph nodes within 12 h of cell transfer and require loading of the dendritic cells with the appropriate peptide antigen. Dendritic cells undergo specific morphologic changes following interactions with antigen-specific T cells.     

Transgenic mice carrying the mouse mammary tumor virus ras fusion gene: distinct effects in various tissues.

Transgenic mice carrying the v-Ha-ras oncogene under the control of the mouse mammary tumor virus long terminal repeat were produced. These mice exhibit several phenotypes: mammary tumors, bilateral hyperplasia of the harderian lacrimal gland, primary bronchio-alveolar lung adenocarcinoma, and splenomegaly. High levels of the transgene RNA were detected in mammary, harderian, and lung tumors. Accumulation of cells of the myeloid lineages was found in enlarged spleens. This phenotype may represent an indirect effect of v-Ha-ras expression on myeloid progenitors. Our data illustrate the cell-specific effects of v-Ha-ras.     

Cellular gene expression in papillomas of the choroid plexus from transgenic mice that express the simian virus 40 large T antigen.

Transgenic mice that contain the simian virus 40 (SV40) enhancer-promoter and large tumor (T) antigen gene develop papillomas of the choroid plexus. The tumors remain well differentiated on histological examination and express normal levels of tissue-specific mRNAs for transthyretin (TTR) and the 5-HT1C serotonin receptor, two differentiated cell markers. Both Northern (RNA) blot analysis and in situ cytohybridization have been used to monitor the steady-state levels of the mRNAs from the viral oncogene (T antigen) and from several cellular oncogenes. In situ hybridization demonstrated, in serial sections, increased levels of both T antigen mRNA and p53 mRNA localized in the tumor tissue but not in the normal brain tissue. The ratios of the steady-state levels of mRNA for p53/TTR and p53/L32, a ribosomal protein gene, were 2- to 20-fold higher in the tumor tissue than in the normal choroid plexus tissue. Several other oncogenes did not show elevated levels of mRNA in these tumors. p53 protein levels were not detectable in normal brain tissue, but p53 levels were very high in tumor tissue in which all of the p53 was found in a complex with the SV40 large T antigen. These data continue to show a close relationship between SV40 T-antigen-mediated tumorigenesis and the role of p53 in these tumors.     

Requirement for Both Shc and Phosphatidylinositol 3′ Kinase Signaling Pathways in Polyomavirus Middle T-Mediated Mammary Tumorigenesis

Transgenic mice expressing the polyomavirus (PyV) middle T antigen (MT) develop multifocal mammary tumors which frequently metastasize to the lung. The potent transforming activity of PyV MT is correlated with its capacity to activate and associate with a number of signaling molecules, including the Src family tyrosine kinases, the 85-kDa Src homology 2 subunit of the phosphatidylinositol 3′ (PI-3′) kinase, and the Shc adapter protein. To uncover the role of these signaling proteins in MT-mediated mammary tumorigenesis, we have generated transgenic mice that express mutant PyV MT antigens decoupled from either the Shc or the PI-3′ kinase signaling pathway. In contrast to the rapid induction of metastatic mammary tumors observed in the strains expressing wild-type PyV MT, mammary epithelial cell-specific expression of either mutant PyV MT resulted in the induction of extensive mammary epithelial hyperplasias. The mammary epithelial hyperplasias expressing the mutant PyV MT defective in recruiting the PI-3′ kinase were highly apoptotic, suggesting that recruitment of PI-3′ kinase by MT affects cell survival. Whereas the initial phenotypes observed in both strains were global mammary epithelial hyperplasias, focal mammary tumors eventually arose in all female transgenic mice. Genetic and biochemical analyses of tumorigenesis in the transgenic strains expressing the PyV MT mutant lacking the Shc binding site revealed that a proportion of the metastatic tumors arising in these mice displayed evidence of reversion of the mutant Shc binding site. In contrast, no evidence of reversion of the PI-3′ kinase binding site was noted in tumors derived from the strains expressing the PI-3′ kinase binding site MT mutant. Tumor progression in both mutant strains was further correlated with upregulation of the epidermal growth factor receptor family members which are known to couple to the PI-3′ kinase and Shc signaling pathways. Taken together, these observations suggest that PyV MT-mediated tumorigenesis requires activation of both Shc and PI-3′ kinase, which appear to be required for stimulation of cell proliferation and survival signaling pathways, respectively.     

A mechanism of drug resistance to tamoxifen in breast cancer

Drug resistance to tamoxifen (Tam) is a significant clinical problem but the mechanism through which this occurs remains elusive. We have developed a number of xenograft models of Tam-stimulated growth that model breast cancer progression using estrogen receptor positive MCF-7 or T47D breast cancer cells. When estrogen-stimulated T47D:E2 tumors are treated long term with Tam, Tam-stimulated tumors develop (T47D:Tam) that are stimulated by both estrogen and Tam. When HER-2/neu status is determined, it is clear that the T47D:Tam tumors express significantly higher levels of HER-2/neu protein by immunohistochemistry and mRNA as measured by real-time RT-PCR. The T47D:Tam tumors also express higher levels of estrogen receptor and progesterone receptor protein than their estrogen-stimulated T47D:E2 counterparts. We compared out results to the MCF-7 model of Tam-stimulated growth. The MCF-7:Tam ST (estrogen- and Tam-stimulated) and MCF-7:Tam LT (estrogen-inhibited, Tam-stimulated) were bilaterally transplanted to account for any mouse to mouse variation and characteristic growth patterns were observed. TUNEL staining was performed on MCF-7:Tam LT treated with either estrogen or Tam and it was concluded that estrogen-inhibited tumor growth was a result of increased apoptosis. Three phases of tumor progression are described that involve increases in HER-2/neu expression, de-regulation of estrogen receptor expression and increases in apoptosis which in concert determine the phenotype of drug resistance to Tam.     

Oncogenes result in genomic alterations that activate a transcriptionally silent, dominantly selectable reporter gene (neo).

Although oncogenes and tumor suppressor genes have been implicated in carcinogenesis and tumor progression, their relationship to the development of genomic instability has not been elucidated. To examine this role, we transfected oncogenes (polyomavirus middle [Py] and large T [MT and LT]) and adenovirus serotype 5 E1A) into two NIH 3T3-derived cell lines, EN/NIH 2-4 and EN/NIH 2-20. Both cell lines contain two stable integrants of a variant of the retrovirus vector pZipNeoSV(x)1 that has been modified by deletion of the enhancer elements from the long terminal repeats. DNA rearrangements activating the silent neomycin phosphotransferase gene (neo) present in these integrants were identified by selection of cells in the antibiotic G418. Whereas control-transfected EN/NIH cell lines do not yield G418-resistant subclones (GRSs), a fraction of oncogene-transfected EN/NIH 2-4 (8 of 19 Py MT, 5 of 17 Py LT, and 11 of 19 E1A) and 2-20 (7 of 15 Py MT) cell lines gave rise to GRSs at differing frequencies (0.33 x 10(-6) to 46 x 10(-6) for line 2-4 versus 0.11 x 10(-6) to 1.3 x 10(-6) for line 2-20) independent of cell generation time. In contrast, a distinctly smaller fraction of mutant Py MT-transfected EN/NIH cell lines (1 of 10 MT23, 1 of 10 MT1015, and 0 of 10 MT59b) resulted in GRSs. Southern analysis of DNA from selected oncogene-transfected GRSs demonstrated genomic rearrangements of neo-containing cellular DNA that varied in type (amplification and/or novel fragments) and frequency depending on the specific oncogene and EN/NIH cell line used in transfection. Furthermore, only one of the two neo-containing genomic loci present in both EN/NIH cell lines appeared to be involved in these genomic events. In addition to effects related to the genomic locus, these observations support a role for oncogenes in the development of genetic changes associated with tumor progression.     

Local costimulation reinvigorates tumor-specific cytolytic T lymphocytes for experimental therapy in mice with large tumor burdens

Cytotoxic T cells recognize tumor Ags and destroy cancer cells in vitro. Adoptive transfer studies with transgenic T cells specific for tumor Ags have demonstrated that CTL are effective only in mice with small tumor burdens and thus appear to have limited potential in cancer immunotherapy. Here we used transgenic mice that express the TCR specific for an unmutated tumor Ag P1A and multiple lineages of P1A-expressing tumors to address this critical issue. We found that local costimulation, either by expression of B7-1 on the tumor cells or by local administration of anti-CD28 mAb 37N, reinvigorated the function of CTL specific for the tumor Ag, as it substantially increased the efficacy of CTL therapy for mice with large tumor burdens. Our study suggests that CTL-based immunotherapy can be manipulated to deal with large tumors.     

Consequences of widespread deregulation of the c-myc gene in transgenic mice: multiple neoplasms and normal development

We have constructed a transgenic mouse strain in which a mammary tumor virus LTR/c-myc fusion gene is anomalously expressed in a wide variety of tissues. The deregulated c-myc transgene, now glucocorticoid inducible, contributes to an increased incidence of a variety of tumors, including those of testicular, breast, lymphocytic (B cell and T cell), and mast cell origin. The deregulated gene does not, however, otherwise disturb cell proliferation, nor does it interfere with normal development in these animals. Moreover, since not all tissues that express the transgene develop neoplasms, these results begin to define the transforming spectrum of the c-myc oncogene. They also extend to several organ systems the notion that elements in addition to an activated c-myc gene are required to induce malignancy in the living organism.     

Transgenic mice harboring SV40 t-antigen genes develop characteristic brain tumors

A high percentage of transgenic mice developing from eggs microinjected with plasmids containing the SV40 early region genes and a metallothionein fusion gene develop tumors within the choroid plexus. A line of mice has been established in which nearly every affected animal succumbs to this brain tumor. Thymic hypertrophy and kidney pathology are also observed in some mice. SV40 T-antigen mRNA and protein are readily detected in affected tissues; however, SV40 T-antigen gene expression is barely detectable in unaffected tissues or in susceptible tissues prior to overt pathology, suggesting that tumorigenesis depends upon activation of the SV40 genes. Comparison of DNA from tumor tissue (or cell lines derived from tumors) with DNA from unaffected tissues reveals structural rearrangements as well as changes in DNA methylation of the foreign DNA. The SV40 genes are frequently amplified in tumor tissue, which further indicates that their expression is intimately involved in tumorigenesis in transgenic mice.