SV40 large T antigen with c-Jun down-regulates myelin P0 gene expression: a mechanism for papovaviral T antigen-mediated demyelination

Expression of myelin proteins has been shown to be altered in transgenic mice that express papovaviral large tumor (T) antigens. This paper analyzes the effect on P0 gene expression in secondary Schwann cells transfected with the SV40 T antigen gene and in Schwann cells immortalized by T antigen. In secondary Schwann cells, both T antigen and c-Jun are required for significant inhibition of the P0 promoter; expression of only one of the proteins is insufficient for repression of the P0 gene. T antigen, c-Jun (p39), and c-Jun-related protein (p47) form an immunoprecipitable complex in SV40 immortalized Schwann cell lines, and T antigen and c-Jun bind independently and as a complex to the P0 promoter. Our data suggest that the probable molecular mechanism underlying the hypomyelination observed in transgenic animals expressing T antigen may be due to the repression of the P0 gene by T antigen and c-Jun.     

Migration, differentiation and integration of an immortalized neural cell line transplanted into the neonatal and adult mouse brain

An immortalized neural cell line V1 was transplanted stereotaxically into the cerebellum and hippocampus of developing and adult mice, and the mode of migration, differentiation and arrangement of the grafted cells were examined by labeling the grafted cells with DiI (1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate) and immunohistochemical staining. This cell line was established by transduction of the temperature-sensitive allele tsA58 of SV40 large T antigen oncogene into mouse hypothalamic cells. Grafted cells did not show any tumorigenicity for a long time. Some of the cells grafted into the neonatal cerebellum and hippocampus were arranged along the host cortical layer and showed neuronal or glial differentiation according to the grafted site. The cells grafted into adult cerebellum also showed migration and arrangement along the host cortical layer as well as morphological differentiation into glial cells in a manner similar to that of transplantation to the neonate. On the other hand, the cells grafted into the adult hippocampus made only clusters without forming an organized arrangement. These findings suggest that the grafted cells are integrated into the developmental processes of the host brain, and the mode of differentiation and arrangement of the grafted cells depends on the microenvironment of the different developmental stages of the host brain. The involvement of host blood vessels and astroglial framework in the migration and arrangement of the grafted cells was also suggested. Furthermore, these findings suggest the plasticity of the host brain in response to the grafted cells and the possibility of reconstructing the host brain with this multipotential neural cell line.     

Fenofibrate modifies transaminase gene expression via a peroxisome proliferator activated receptor alpha-dependent pathway

Supporting cells in the inner ear sensory epithelium are most likely hair cell progenitors. In an effort to establish an in vitro model system of hair cell differentiation, we developed immortalized epithelial cell lines by transferring the tsA58 allele of the SV40 large T antigen oncogene into neonatal rat utricular supporting cells using a retrovirus. The established cell lines have been stably maintained continuously for more than 25 passages and display many features similar to primary supporting cells. They grow in patches and assume a polygonal morphology. Immunocytochemical characterization of the established cell lines reveals that these cells can be labeled by epithelial cell markers, but not by fibroblast, glial or neuronal markers. The immortalized cells grow rapidly in serum medium at permissive temperature, but the majority cease proliferation when cultured in serum free medium at non-permissive temperature. These cells respond to mitogenic growth factors including bFGF, EGF and TGF-alpha and express growth factor receptors in a manner similar to the primary supporting cells. Furthermore, we find that the cells undergo a morphological differentiation when cultured in serum free medium at non-permissive temperature in the presence of bFGF. Under these conditions, the cells shrink in size, become elongated, and express early hair cell markers such as calretinin and calmodulin. The utricular epithelial cell line we have established may potentially provide an invaluable system for studying hair cell differentiation and regeneration.     

Conditional immortalization of human endometrial stromal cells with a temperature-sensitive simian virus 40

The study of immortalization and other alterations associated with neoplastic transformation of endometrial stromal cells is important to understanding the development of uterine sarcomas and mixed tumors. Because stromal cells are important regulators of associated epithelial cells, alterations in the regulation of stromal cell proliferation that influence epithelial cells may also contribute to the development of endometrial carcinomas. To study immortalization and associated phenotypic and genetic alterations of human endometrial stromal cells, cultures were transfected with a plasmid containing an ori-, temperature-sensitive mutant SV40, A209 (tsSV40). Morphologically transformed colonies were selected and propagated at the permissive temperature until they entered 'crisis'. In contrast to human fibroblasts, every clone tested was immortalization competent. The frequency of immortalization was approximately 1 x 10(-6). One uncloned and six cloned cell lines escaped from crisis and appear to be immortal. Two clones, M4 and B10T1, were selected for further study. Immortalization is conditional; proliferative arrest occurs at the restrictive temperature for large T antigen function. Furthermore, withdrawal of the large T antigen results in expression of the senescent phenotype of enlarged, flattened cells. Colony-forming efficiency at the restrictive temperature was undetectable. Immortalization is also associated with several genetic alterations. The DNA content of tsSV40 transfected cells was either diploid or tetraploid in the precrisis stage of proliferation, but became aneuploid upon immortalization. Several structural rearrangements of chromosomes were detected in the immortalized stromal cells which differ from those found in SV40 immortalized fibroblasts. Although their capacity for anchorage-independent proliferation (AIP) is variable, tsSV40-immortalized endometrial stromal cells have a higher capacity for AIP than their tsSV40-transfected progenitor cells in the period of proliferation prior to 'crisis'.     

SV40 T antigen directed by a powerful erythroid enhancer-promoter produced sarcomas and pancreatic tumors but not erythroid-specific tumors in transgenic mice

We have expressed the simian virus 40 (SV40) large T antigen oncogene in erythroid tissues of mice to test its ability to immortilize erythroid cells. A transgene construct was built in which the SV40 large T antigen structural gene was linked to erythroid-specific enhancer and promoter sequences. The enhancer employed was the human beta-globin family microlocus control region, and the promoter sequences were derived from the human beta-globin promoter. Transgenic mice were generated and they expressed T antigen in the bone marrow and spleen cells. Yet, no hematopoietic neoplasia arose in these mice. Instead, after a lag period of 2-6 months, the mice developed soft tissue sarcomas and pancreatic islet-cell tumors that expressed high levels of T antigen.     

Establishment and neurite outgrowth properties of neonatal and adult rat olfactory bulb glial cell lines

Two glial cell types surround olfactory axons and glomeruli in the olfactory bulb (OB) and may influence synapse development and regeneration. OB astrocytes resemble type-1 astrocytes, and OB ensheathing cells resemble non-myelinating Schwann cells. We have produced clonal OB astrocyte and ensheathing cell lines from rat neonatal and adult OB cultures by SV40 large T antigen transduction. These cell lines have been characterized by morphology, growth characteristics, immunophenotype, and ability to promote neurite outgrowth in vitro. Neonatal and adult ensheathing cell lines were found to support higher neurite outgrowth than OB astrocyte lines. Neonatal OB astrocyte lines were of two types, high and low outgrowth support. The low support astrocyte lines express J1 and a chondroitin sulfate-containing proteoglycan as do astrocytes encircling the neonatal glomeruli in vivo. The adult OB astrocyte cell lines supported lower levels of outgrowth than adult ensheathing cell lines. These results are consistent with a positive role for ensheathing cells in OB synapse regeneration, in vivo. Further, based on our results, we hypothesize that ensheathing cells and high-outgrowth astrocytes facilitate axon growth in vivo, while low outgrowth astrocytes inhibit axon growth and may facilitate glomerulus formation.     

In vitro and in vivo analysis of a rat bipotential O-2A progenitor cell line containing the temperature-sensitive mutant gene of the SV40 large T antigen

Primary cultures from neonatal optic nerve contain pluripotential O-2A progenitor cells that are capable of differentiating into oligodendrocytes, type-2 astrocytes or adult O-2A progenitors (O-2Aadult). Since primary optic nerve cultures contain a mixture of glial cell types of which only a small number are O-2A progenitors, experiments on cell lineage and differentiation carried out using these cultures are both intrinsically limited and difficult to interpret. Ideally, cells from a clonal cell population would provide the optimal starting material for biological studies. In this paper we describe the creation of an O-2A progenitor cell line using a retrovirus carrying a temperature-sensitive mutant SV40 large T antigen gene. This cell line has provided sufficient numbers of cells to allow analysis of their in vitro properties and their behaviour following transplantation into an in vivo environment. At the non-permissive temperature (39 degrees C), these cells differentiate into oligodendrocytes and type-2 astrocytes in a similar fashion to O-2A progenitor cells from primary cultures (O-2Aprim). When grown in media containing platelet-derived growth factor and basic fibroblast growth factor, the cell numbers can be expanded in culture without differentiating, consistent with the behaviour of O-2Aprim progenitor cells. By exploiting this property, it has been possible to culture large numbers of O-2A progenitors for in vivo analysis. In this study we have shown that transplantation of this O-2A cell line into glia-free areas in adult rat spinal cord results in differentiation of a proportion of cells into oligodendrocytes which are capable of myelinating axons. Furthermore, differentiation of O-2A cells into astrocytes was also observed, indicating that the bipotentiality of these cells in vitro can also be demonstrated in vivo.     

N-stearoyl-phosphatidylserine: synthesis and role in divalent-cation-induced aggregation and fusion

Ex vivo gene therapy, in which hepatocytes are harvested from mutants, retrovirally transduced with a normal gene and transplanted back into the donor, has been used for correction of inherited metabolic defects of liver. Major drawbacks of this method include limited availability of autologous hepatocytes, inefficient retroviral transduction of primary hepatocytes, and the limited number of hepatocytes that can be transplanted safely. To obviate these problems, we transduced primary hepatocytes derived from inbred bilirubin-UDP-glucuronosyl-transferase (BUGT)-deficient Gunn rats by infection with a recombinant retrovirus expressing temperature-sensitive mutant SV40 large T antigen (tsT). The immortalized cells were then transduced with a second recombinant retrovirus expressing human B-UGT, and a clone expressing high levels of the enzyme was expanded by culturing at permissive temperature (33 degrees C). At 37 degrees C, tsT antigen was degraded and the cells expressed UGT activity toward bilirubin at a level approximately twice that present in normal rat liver homogenates. For seeding the cells into the liver bed, 1 x 10(7) cells were injected into the spleens of syngeneic Gunn rats five times at 10-day intervals. Excretion of bilirubin glucuronides in bile was demonstrated by HPLC analysis and serum bilirubin levels were reduced by 27 to 52% in 40 days after the first transplantation and remained so throughout the duration of the study (120 days). None of the transplanted Gunn rats or SCID mice transplanted with the immortalized cells developed tumors.     

Peptide-induced T cell clones: specificity, MHC restriction, proliferation and cytokine pattern as a function of different stimulations

CD4+ T cell clones were generated to tetanus toxin or to two tetanus toxin-derived peptides p2 (AA 830-834) and p30 (AA 947-976). 11 of the 24 p30-specific clones reacted to shorter p30 subunits (p301 or p302), and only 14 of the p2 or p30-specific clones reacted with TT presented by EBV-transformed B cell lines (B-LCL). The p30-specific clones were HLA-DP4 restricted. In contrast to autologous B cell lines, the majority of allogeneic, but HLA-DP4-positive cell lines failed to present p30 to the specific clones. We concluded that T cell clones are highly specific and that both, small alterations of the peptide length as well as discrete differences of the HLA-molecule may abrogate recognition of the peptide HLA complex by T cells. Moreover, use of peptides as stimulators of T cells may recruit and activate T cells which fail the "original" peptide, derived from normal antigen processing. Clones could usually be maintained in culture for 4-6 months, but with the help of freezing and thawing some clones are now available for over 2 years and still specific. Comparison of different autologous antigen-presenting cells, namely B-LCL and activated MHC class II-positive T cells revealed that not all clones were able to mount a proliferative response to peptide presentation by T cells, while all clones proliferated to B cells as APC. If stimulated with peptide and B-LCL, the clone proliferating to T cells as APC (so-called T responder clones) secreted a broad spectrum of cytokines (Th0-like) and were easier to maintain in culture. In contrast, clones which were unable to proliferate to peptide presentation, so-called T-nonresponder clones, showed a more restricted cytokine pattern and elevated or very low IL4/IFN gamma ratio upon antigen specific stimulation. However, all clones secreted at least small amounts of IL2, IL4, IFN gamma and TNF alpha, if stimulated by PMA and ionomycin. Thus, both chemical and antigen-specific stimulations should be considered if T cell clones are classified as Th1 or Th2, whereby those clones, which secrete a limited cytokine pattern after antigen stimulation only, might be named Th1 or Th2 like clones, while clones which even after PMA/ionomycin do not secrete all cytokines, might represent "real" Th1 or Th2 clones.     

The morphological characteristics of a papillomavirus infection in the epithelial tissue of the cervix uteri in comparison with the data from the molecular biological identification of specific HPV DNA sequences]

Xeroderma pigmentosum group E (XP-E) fibroblasts (XP95TO) were transformed with pSV3neo. Selection in medium containing G418 yielded 14 clones with extended life span. Following crisis, one clone was recovered that behaved in culture as an immortal cell line and was named XPET6/1. Expression of the SV40 large T antigen gene (Tag) and increased level of p53 were demonstrated by western analyses. Fingerprinting with 14 polymorphic microsatellite genetic markers confirmed that XPET6/1 originated from the parental strain XP95TO. XPET6/1 retained the sensitivity to killing by UV observed with the parental strain. Cell-free extracts from the immortal or the parental XP-E cells were deficient in excision, compared to extracts from HeLa or extracts from Tag-transformed XP variant fibroblasts. Complementation of XP-E extracts with XP-A, XP-D or XP-G extracts restored nucleotide excision activity to normal levels. XPET6/1 could prove a useful cell line for cloning of the XPE gene by functional complementation.     

Passive but not active CD8+ T cell-based immunotherapy interferes with liver tumor progression in a transgenic mouse model

To evaluate tumor immunotherapies, we used transgenic mice that harbor a progressive liver tumor associated with the expression of the SV40 large tumor T oncoprotein (SV40-T). To induce "self" tumor Ag-specific CD8+ T cells, mice were injected with an immunodominant SV40-T CTL epitope mixed with a heterologous helper peptide. Despite repeated injections, this vaccine failed to raise a tumor-specific CD8+ T cell response that was efficient enough to counteract tumors. Although coimmunization with SV40-T CTL epitope and heterologous helper peptide efficiently recruited the respective Th cells, only low-avidity SV40-T-specific CD8+ T cells were activated. Furthermore, major alterations in SV40-T-specific B and Th cell responses were characterized. In contrast, transfers of higher-avidity CTLs specific for the same SV40-T epitope were effective in counteracting tumors. These results suggest that passive therapies targeted to self tumor Ag may be more suitable than active immunization in the treatment of spontaneous tumors.     

Inhibition of antigen-induced muscle contractions by inhibitors of thromboxane pathway in rat small intestine

The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (CDK) in association with cyclins. In mammalian cells the timing of activation of cyclin A-associated kinase activity coincides with the onset of DNA synthesis in S-phase. Using in vitro replication of SV40 origin-containing DNA as a model system, we have analyzed the proteins associated with DNA during initiation of DNA replication in S-phase cell extracts. This analysis reveals that, in addition to replication initiation proteins, cyclin A and cdk2 are also specifically associated with DNA. The association of cyclin A and cdk2 with DNA during initiation is cell cycle regulated and occurs specifically in the presence of SV40 origin-containing plasmid and SV40 T antigen (the viral replication initiator protein). The interactions among proteins involved in initiation play an important role in DNA replication. We therefore investigated the ability of cyclin A and cdk2 to associate with replication initiation proteins. Under replication initiation conditions, cyclin A and cdk2 from S-phase extracts specifically associate with SV40 T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinant cyclin A associates directly with SV40 T antigen. Taken together, our results suggest that cyclin A and cdk2 are components of the SV40 replication initiation complex, and that protein-protein interactions between cyclin A-cdk2 and T antigen may facilitate the association of cyclin A-cdk2 with the complex.     

Efficient production of JC virus in SVG cells and the use of purified viral antigens for analysis of specific humoral and cellular immune response

A new in vitro system for the production of the human polyomavirus JC virus (JCV) was established to circumvent the need for virus growth in primary human fetal glial cells (PHFG). The permanent cell line SVG, transformed by an origin-defective mutant of Simian Virus 40 (SV40) was used to grow JCV. JCV-specific RNA could be detected at day 5 and viral antigen at day 6 post infection (p.i.). Virus production peaked at day 16. Virus could be purified by differential centrifugation. The purified fraction consisted mainly of mature particles but contained also pentamers of the major structural virus protein 1 (VP1). The VP1-pentamers could be purified to near homogeneity. The purified virus particles stimulated a specific T-cell proliferation of peripheral blood monocytes (PBMCs) of a patient with progressive multifocal leukoencephalopathy (PML) and of two healthy individuals. In addition, JCV-particles and VP1-pentamers reacted specifically in an ELISA with a series of five PML-patient sera and four sera of individuals not affected by PML. These results demonstrate that purified whole virus particles are suitable for the analysis of specific cellular and humoral immune responses to JCV.     

Disregulation of mitotic checkpoints and regulatory proteins following acute expression of SV40 large T antigen in diploid human cells

SV40 large T antigen (T) inactivates the tumor suppressor proteins p53 and pRb, and can induce cells to enter DNA replication at inappropriate times. We show here that T also compromises three cell cycle checkpoints that regulate the entry into and exit from mitosis. Human diploid fibroblasts infected with a retrovirus expressing T displayed an attenuated radiation-induced mitotic delay, were more susceptible to chemical-induced uncoupling of mitosis from the completion of DNA replication, and were more likely to exit mitosis and rereplicate their DNA when mitotic spindle assembly was inhibited. Consistent with altered mitotic checkpoint control, cells expressing T displayed elevated protein levels and/or associated activities of the mitotic regulatory proteins cyclin A, cyclin B, Cdc25C and p34(cdc2). These changes in mitotic control were evident within 5-10 population doublings after retroviral infection, indicating a direct effect of T expression. Cells acutely infected with the T-expressing retrovirus suffered numerical and structural chromosome aberrations, including increases in aneuploidy, dicentric chromosomes, chromatid exchanges and chromosome breaks and gaps. These findings indicate that T rapidly disrupts mitotic checkpoints that help maintain genomic stability, and suggest mechanisms by which T induces chromosome aberrations and promotes the immortalization and neoplastic transformation of human cells.     

Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier

The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood-brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3-neo coding for the SV40 large T antigen and the neomycin gene. The neomycin-resistant transfected cells overcame proliferative senescence, and after a 6-8 wk period of crisis produced immortalization-competent cell colonies. Single-cell clones of near-diploid genotype were isolated from these colonies, propagated, and characterized. Immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates, but remained serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV-HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control cells including expression of factor VIII-related antigen, uptake of acetylated low-density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor-mediated endocytosis, and high activities of the BBB-specific enzymes alkaline phosphatase and gamma-glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV-HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV-HCEC monolayers by 2.5-fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.     

Role of a subdominant H-2Kd-restricted SV40 tumor antigen cytotoxic T lymphocyte epitope in tumor rejection

SV40-transformed mKSA cells (H-2d) readily induce progressively growing tumors in adult syngeneic BALB/c mice while expressing the full complement of H-2d MHC class I antigens. BALB/c mice previously immunized with SV40, soluble SV40 T antigen, or irradiated SV40-transformed syngeneic, allogeneic, or xenogeneic cells reject an mKSA tumor challenge even though these mice have been considered low- or nonresponders to T antigen due to difficulty in demonstrating SV40 T antigen-specific CTL. We have investigated the role of H-2d-restricted CTL in the rejection of SV40 tumors in BALB/c mice. Immunization of BALB/c mice with SV40 induced T antigen-specific CTL which were largely. H-2Ld-restricted. However, following repeated in vitro restimulation with mKSA cells, CTL emerged which recognized a subdominant H-2Kd-restricted epitope corresponding to T antigen residues 499-507. Immunization of BALB/c mice with a recombinant vaccinia virus expressing the T499-507 epitope provided partial protection against a challenge of syngeneic mKSA tumor cells and induced the generation of T499-507-specific CTL. These results indicate that a subdominant H-2Kd-restricted CTL epitope can participate in the rejection of SV40 tumors in BALB/c mice.     

Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA

All animal DNA viruses except pox virus utilize the cell nucleus as the site for virus reproduction. Yet, a critical viral infection process, nuclear targeting of the viral genome, is poorly understood. The role of capsid proteins in nuclear targeting of simian virus 40 (SV40) DNA, which is assessed by the nuclear accumulation of large tumor (T) antigen, the initial sign of the infectious process, was tested by two independent approaches: antibody interception experiments and reconstitution experiments. When antibody against viral capsid protein Vp1 or Vp3 was introduced into the cytoplasm, the nuclear accumulation of T antigen was not observed in cells either infected or cytoplasmically injected with virion. Nuclearly introduced anti-Vp3 IgG also showed the inhibitory effect. In the reconstitution experiments, SV40 DNA was allowed to interact with protein components of the virus, either empty particles or histones, and the resulting complexes were tested for the capability of protein components to target the DNA to the nucleus from cytoplasm as effectively as the targeting of DNA in the mature virion. In cells injected with empty particle-DNA, but not in minichromosome-injected cells, T antigen was observed as effectively as in SV40-injected cells. These results demonstrate that SV40 capsid proteins can facilitate transport of SV40 DNA into the nucleus and indicate that Vp3, one of the capsid proteins, accompanies SV40 DNA as it enters the nucleus during virus infection.     

Reduced growth factor requirements and accelerated cell-cycle kinetics in adult human melanocytes transformed with SV40 large T antigen

Melanomas develop with high frequency in transgenic mice in which oncogenic sequences of the SV40 DNA tumor virus have been specifically targeted to melanocytes. To investigate the role of SV40 in melanomagenesis, cultured human melanocytes were transformed with a retroviral shuttle vector encoding the SV40 large T antigen and examined for changes in cell-cycle kinetics and growth-factor dependence. Colonies expressing the viral oncogene were morphologically indistinguishable from their non-T-antigen-transformed counterparts. Also like normal melanocytes, the infected cells remained anchorage dependent and non-tumorigenic in nude mice. However, T-antigen-positive cultures exhibited significantly accelerated population doubling times, increased saturation densities with highly confluent monolayers and a three- to fourfold extended life span. Most interestingly, cell-cycle analysis revealed a measurable shift from quiescent to cycling cells in T-antigen-expressing cultures and an acquired ability to progress more rapidly through G1. Moreover, T-antigen-positive melanocytes proliferated in the absence of PMA and required markedly reduced levels of exogenous bFGF. These studies indicate that the viral oncogen of simian virus 40 provides melanocytes with distinct growth advantages that may render these cells unusually susceptible to additional environmental challenges necessary for full expression of the malignant phenotype.