Embryonic lethalities and endothelial tumors in chimeric mice expressing polyoma virus middle T oncogene

The effect of the middle T oncogene of polyoma virus was studied in vivo using a replication-defective selectable retrovirus. Injection of virus into newborn and adult mice resulted in the rapid appearance of cavernous hemangiomas. Infection of embryos did not yield transgenic mice; therefore, embryonal stem (ES) cells were used as an alternative system. Several infected ES cell clones were established that constitutively expressed middle T and its associated tyrosine kinase activity. Chimeric embryos obtained by blastocyst injection of individual ES cell clones were specifically arrested at midgestation, when multiple hemangiomas disrupted blood vessel formation. From these tumors endothelial cell lines were established that retained expression of von Willebrand factor yet were tumorigenic in vivo. These results suggest that middle T acts in endothelial cells as a single-step oncogene and that ES cells provide a valuable system for the study of growth control during embryogenesis.     

Isolation and characterization of an established endothelial cell line from transgenic mouse hemangiomas

A murine endothelial cell line was isolated from hemangiomas induced by expression of the polyoma early region gene in transgenic mice. After two cell sortings using acetylated low-density lipoprotein with a fluorescent label (Dil-Ac-LDL), a pure population of endothelial cells has been carried for more than 60 passages from the animal. The cells retain endothelial cell properties such as a characteristic cobblestone appearance at confluency, contact-inhibited growth, and active uptake of Ac-LDL. Expression analysis shows that the cells express both the polyoma transgene and the von Willebrand factor, an endothelial cell marker. Subcutaneous injection of the cultured endothelial cells into nontransgenic histocompatible mice or nude mice led to hemangioma formation, and endothelial cells were re-isolated by cell sorting from these secondary hemangiomas. This cell line represents a renewable source of murine endothelial cells derived from transgenic mice that can be studied both in vitro and by reintroduction into a host.     

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.     

Polyomavirus middle T antigen induces ribosomal protein S6 phosphorylation through pp60c-src-dependent and -independent pathways.

Phosphorylation of ribosomal protein S6 is elevated in polyomavirus-infected cells. This elevation results only in part from activation of S6 kinase activity. These effects appear to reflect independent activities of wild-type middle T antigen. Hr-t mutant NG59, encoding a defective middle T protein, and mutant Py808A, encoding no middle T protein, were unable to induce S6 kinase activity or elevate S6 phosphorylation. Two other site-directed mutants encoding altered middle T proteins did elevate S6 phosphorylation while only weakly stimulating S6 kinase activity. These results suggest at least two independent pathways leading to elevation of S6 phosphorylation. One pathway leads to induction of S6 kinase activity following activation of pp60c-src by transformation-competent middle T antigen. Another pathway operates independently of S6 kinase induction and can be regulated by transformation-defective middle T mutants such as Py1387T. This mutant, encoding a truncated middle T protein that failed to associate with the plasma membrane and to activate pp60c-src, caused increased levels of S6 phosphorylation without detectably increasing S6 kinase activity. The ability of mutants such as Py1387T to induce S6 phosphorylation correlated with their ability to increase phosphorylation of VP1, an event linked to maturation of infectious virions.     

Protein kinase activity associated with polyoma virus middle T antigen in vitro.

A protein kinase activity can be detected in immunoprecipitates of extracts from polyoma virus (Py)-infected cells using antiserum raised against Py-transformed cells (anti-T serum). The activity is not detected in uninfected cells or when using control serum. Using rat anti-T serum both Py middle T and the heavy chain of rat IgG are phosphorylated, whereas using hamster anti-T serum only Py middle T is phosphorylated. Experiments using a number of different mutants of Py indicate that the kinase activity detected is under viral control and is associated with Py middle T. Consistent with this the kinase, like middle T, can be detected in purified preparations of plasma membranes. The kinase can also be detected in a large number of Py-transformed cells, but not in untransformed cells or in cells transformed by other viruses. Some of the Pytransformed cells which contain kinase activity lack full sized Py large T but all contain middle T. Kinase activity is not detected in a cell line (18.37) which contains integrated viral DNA of a nontransforming hr-t deletion mutant and which contains Py large T but not middle T or small t. These results show that Py middle T or a protein which specifically binds to it has protein kinase activity in vitro. Although these results raise the possibility that protein kinases play an essential role in Py-induced transformation, however, thus far we have no data which show unequivocally that the results are physiologically significant.     

Endothelioma cells expressing the polyoma middle T oncogene induce hemangiomas by host cell recruitment

Mouse endothelioma cells expressing the polyoma middle T oncogene induced hemangiomas in a variety of species such as mice, rats, chicks, and quails. In embryos and newborn mice the hemangiomas expanded within 10-18 hr of injection, disrupting the vasculature and causing the death of the animal. In contrast, the hemangiomas formed a stable structure reminiscent of benign human hemangiomas in adult mice within 5 days. Analysis of the cells comprising the hemangioma revealed that over 95% of the endothelial cells were host derived. No induction of host cell proliferation was detected, and no endothelial mitogens were secreted by the endothelioma cells in vitro. The maintenance of the hemangioma appeared to require the continuous presence of endothelioma cells. The results indicate that these endothelioma cells act as a potent stimulating agent in the rapid formation of hemangiomas by recruiting nonproliferating host endothelial cells.     

The N terminus of hamster polyomavirus middle T antigen carries a determinant for specific activation of p59c-Fyn.

Transformation by rodent polyomaviruses is mediated primarily by middle T antigen, a membrane-bound protein that does not carry an intrinsic enzymatic activity but interacts and subverts the activity of cellular regulators of proliferation. The multiple protein partners of murine polyomavirus (Py) middle T antigen include the tyrosine kinases c-Src and, to a lesser extent, c-Fyn and c-Yes. By contrast, the hamster polyomavirus (HaPV) middle T antigen selectively activates the c-Fyn gene product. This difference may account for the contrasting tumor patterns induced by the two viruses. The sequences of the respective N-terminal and C-terminal functional domains of murine Py and HaPV middle T antigens are highly conserved whereas the intervening stretches are clearly divergent, leading to the speculation that this divergence may direct the specificity for tyrosine kinase activation. We have addressed this issue by constructing a chimera middle T antigen molecule carrying the N-terminal domain from HaPV (exon 1) in phase with the other two domains from murine Py (exon 2). The biological properties of this chimera molecule are indistinguishable from those of HaPV middle T antigen; it specifically activates p59c-Fyn and carries the transforming phenotype of the HaPV middle T antigen on rat fibroblasts.     

Isolation of rat Schwann cell lines: use of SV40 T antigen gene regulated by synthetic metallothionein promoters

Synthetic promoter elements from the mouse metallothionein-I promoter controlling the expression of SV40 T antigen have been tested for their efficacy in cloning rat Schwann cell lines that retained the characteristic properties of these cells and could be passed in culture indefinitely. The constructed promoters contained either four (MT4) or five (MT5) copies of a metal regulatory element 5' to the CAAT and TATA elements from the HSV-1 thymidine kinase gene. Characterization of these promoters in transient expression assays and transformation assays showed that both MT5 and MT4 were approximately 10-fold and 15-fold, respectively, weaker than the wild-type MT-I promoter in the presence of heavy metal inducer. However, in the absence of inducer, the basal activity of both MT5 and MT4 was barely detectable and much lower than that of MT-I. Schwann cells were transfected with plasmids containing the SV40 T antigen gene under the control of the different metallothionein promoters and cell lines were established from each. Only with the MT5 and MT4 promoters were lines obtained that resembled secondary Schwann cells in culture in their morphology, generation time, and demonstration of contact inhibition. In the presence of zinc, the expression of T antigen in the lines derived with MT5 and MT4 was about 10-fold lower than that derived with MT-I. On removal of the inducer this level was reduced, and in one cell line T antigen was undetectable. Concomitant with the reduction in T antigen expression there was an increased expression of Po, a protein specific to myelin-forming Schwann cells, and a decreased expression of glial fibrillary acidic protein, a protein expressed only in nonmyelin-forming Schwann cells. These cell lines, therefore, closely resemble untransfected Schwann cells in culture.     

HAGE,a cancer/testis antigen with potential for melanoma immunotherapy: identification of several MHC class I/II HAGE-derived immunogenic peptides

There remains a need to identify novel epitopes of potential tumour target antigens for use in immunotherapy of cancer. Here, several melanoma tissues and cell lines but not normal tissues were found to overexpress the cancer-testis antigen HAGE at the mRNA and protein level. We identified a HAGE-derived 15-mer peptide containing a shorter predicted MHC class I-binding sequence within a class II-binding sequence. However, only the longer peptide was found to be both endogenously processed and immunogenic for T cells in transgenic mice in vivo, as well as for human T cells in vitro. A different class I-binding peptide, not contained within a longer class II sequence, was subsequently found to be both immunogenic and endogenously processed in transgenic mice, as was a second class II epitope. These novel HAGE-derived epitopes may contribute to the range of immunotherapeutic targets for use in cancer vaccination programs.     

Endothelial cell overexpression of fas ligand attenuates ischemia-reperfusion injury in the heart

Fas ligand (FasL) is a member of tumor necrosis factor family that induces apoptosis in target cells that express Fas. The function of FasL during inflammation remains controversial. In this study, we examined the role of vascular endothelial FasL during acute myocardial ischemia-reperfusion that is closely associated with inflammation. Transgenic mouse lines were established that overexpress human FasL on endothelium under the control of the vascular endothelial cadherin promoter. Expression of FasL transgene was detected at both mRNA and protein levels, and functional transgene-encoded FasL protein was specifically expressed on the surface of vascular endothelial cells. Transgenic mice developed normally and had normal hearts. When subjected to 30 min of myocardial ischemia and 72 h of reperfusion, myocardial infarct size was reduced by 42% in the transgenic mice compared with nontransgenic littermates (p < 0.05). Moreover, hemodynamic data demonstrated that transgenic hearts performed better following ischemia and reperfusion compared with nontransgenic hearts. Myocardial neutrophil infiltration was reduced by 54% after 6 h of reperfusion in transgenic hearts (p < 0.01). Neutrophil depletion prior to ischemia-reperfusion injury led to smaller infarcts that were not different between transgenic and nontransgenic mice, suggesting that endothelial FasL may attenuate ischemia-reperfusion injury by abating the inflammatory response. These results indicate that vascular endothelial FasL may exert potent anti-inflammatory actions in the setting of myocardial ischemia-reperfusion injury.     

An inducible eukaryotic host-vector expression system: amplification of genes under the control of the polyoma late promoter in a cell line producing a thermolabile large T antigen

We have taken advantage of the inherent instability of integrated polyoma (Py) DNA sequences in the presence of a functional viral large T antigen (LT) to develop a eukaryotic host-vector system where copy number is controlled by temperature. A mouse cell line WOP32-4, that constitutively expresses a temperature sensitive (ts) LT, was transfected with plasmids containing the Py origin of DNA replication (ori) and either a neomycin-resistance gene (neo) or chloramphenicol acetyl transferase gene (cat) linked to the Py late promoter. Stable transformants were selected at 39 degrees C, the non-permissive temperature for the ts LT function. Upon shift to 33 degrees C, the resident Py sequences present in the WOP32-4 cells cannot excise due to an ori deletion. However, excision of the transfected plasmid molecules and subsequent extrachromosomal replication occur at high rates leading in some cases to the production of 1000-2000 copies per cell (average) of the plasmid. Proportional increases in either neo-specific mRNA or CAT activity were also observed. In situ hybridization for one cell line indicated that about 20% of temperature-shifted cells contained amplified plasmid DNA.     

Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter

Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.     

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses theInt-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.