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Human adenovirus (HAdV) vectors are promising tools for cancer therapy, but the shortage of efficient animal models for productive HAdV infections has restricted the evaluation of systemic effects to mainly immunodeficient mice. Previously, we reported a highly efficient replication of HAdV-2 in a non-tumorigenic mouse mammary epithelial cell line, NMuMG. Here we show that HAdV-2 gene expression and progeny formation in NMuMG cells transformed with the SV40 T antigen (NMuMG-T cells) were as efficient as in the parental NMuMG cells. Injection of HAdV-2 into tumours established by NMuMG-T in SCID mice caused reduced tumour growth and signs of intratumoural lesions. HAdV-2 replicated within the NMuMG-T-established tumours, but not in interspersed host-derived tissues within the tumours. The specific infection of NMuMG-T-derived tumours was verified by the lack of viral DNA in kidney, lung or spleen although low levels of viral DNA was occasionally found in liver., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Gap junctions are channels that connect the cytoplasm of adjacent cells. Oncogenes such as the middle Tumor antigen of polyoma virus (mT) are known to suppress gap junctional, intercellular communication (GJIC). mT associates with and is tyrosine-phosphorylated by cSrc family members. Specific mT phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) or the SH2 domain of the regulatory subunit of the phosphatidylinositol-3 kinase (PI3k, mT-tyr315). Binding results in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI3 kinase/Akt, respectively, both of which are needed for full neoplastic transformation. To examine the effect of mT-initiated pathways upon gap junctional communication, GJIC was quantitated in rat liver epithelial T51B cells expressing mT-mutants, using a novel technique of in situ electroporation. The results demonstrate for the first time that, although even low levels of wild-type mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr-250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI3k pathway is not required for GJIC suppression, indicating that GJIC suppression is independent of full neoplastic conversion and the concomitant morphological changes. Interestingly, expression of a constitutively active, myristylated form of the catalytic subunit of PI3k, p110, or the constitutively active mutants E545K and H1047R increased GJIC, while pharmacological inhibition of PI3k eliminated communication. Therefore, although PI3k is growth promoting and in an activated form it can act as an oncogene, it actually plays a positive role upon gap junctional, intercellular communication., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pancreatic stellate cells (PSCs) play a key role in the dense desmoplastic stroma associated with pancreatic ductal adenocarcinoma. Studies on human PSCs have been minimal due to difficulty in maintaining primary PSC in culture. We have generated the first conditionally immortalized human non-tumor (NPSC) and tumor-derived (TPSC) pancreatic stellate cells via transformation with the temperature-sensitive SV40 large T antigen and human telomerase (hTERT). These cells proliferate at 33°C. After transfer to 37°C, the SV40LT is switched off and the cells regain their primary PSC phenotype and growth characteristics. NPSC contained cytoplasmic vitamin A-storing lipid droplets, while both NPSC and TPSC expressed the characteristic markers αSMA, vimentin, desmin and GFAP. Proteome array analysis revealed that of the 55 evaluated proteins, 27 (49%) were upregulated ≥3-fold in TPSC compared to NPSC, including uPA, pentraxin-3, endoglin and endothelin-1. Two insulin-like growth factor binding proteins (IGFBPs) were inversely expressed. Although discordant IGFBP-2 and IGFBP-3 levels, IGF-I was found to stimulate proliferation of both NPSC and TPSC. Both basal and IGF-I stimulated motility was significantly enhanced in TPSC compared to NPSC. In conclusion, these cells provide a unique resource that will facilitate further study of the active stroma compartment associated with pancreatic cancer., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The discovery of a number of viruses with the ability to induce tumours in animals and transform human cells has vastly impacted cancer research. Much of what is known about tumorigenesis today regarding tumour drivers and tumour suppressors has been discovered through experiments using viruses. The SV40 virus has proven extremely successful in generating transgenic models of many human cancer types and this review provides an overview of these models and seeks to give evidence as to their relevance in this modern era of personalised medicine and technological advancements., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Disease-cell models that recapitulate specific molecular phenotypes are essential for the investigation of molecular pathogenesis of neurodegenerative diseases including lysosomal storage diseases (LSDs) with predominant neurological manifestations. Herein we report the development and characterization of a cell model for a rapid neurodegenerative LSDs, globoid-cell leukodystrophy (GLD), mostly known as Krabbe disease. GLD is caused by the deficiency of β-galactocerebrosidase (GALC), a lysosomal enzyme that hydrolyzes two glycosphingolipids, psychosine and galactosylceramide. Unfortunately, the available culture fibroblasts from GLD patients consist of a limited research tool as these cells fail to accumulate psychosine, the central pathogenic glycosphingolipid in this LSD that results in severe demyelination. Firstly, we obtained brain samples from the Twitcher (Twi) mice (GALC(twi/twi)), the natural mouse model with GALC deficiency. We immortalized the primary neuroglial cultured cells with SV40 large T antigen, generating the 145M-Twi and the 145C-Wt cell lines from the Twi and control mice, respectively. Both cell lines expressed specific oligodendrocyte markers including A2B5 and GalC. The 145M-Twi cells showed biochemical and cellular disturbances related to GLD neuropathogenesis including remarkable caspase-3 activation, release of cytochrome C into the cytosol and expansion of the lysosomal compartment. Under treatment with glycosphingolipids, 145M-Twi cells showed increased LC3B levels, a marker of autophagy. Using the LC-MS/MS method that we developed, the 145M-Twi cells showed significantly higher levels of psychosine. The 145M-Twi and 145C-Wt lines allowed the development of a robust throughput LC-MS/MS assay to measure cellular psychosine levels. In this throughput assay, l-cycloserine showed to significantly reduce the 145M-Twi cellular levels of psychosine. The established 145M-Twi cells are powerful research tools to investigate the neurologically relevant pathogenic pathways as well as to develop primary screening assays for the identification of therapeutic agents for GLD and potentially other glycosphingolipid disorders., (© 2013.)

The review recounts the history of how the study of the DNA tumor viruses including polyoma, SV40 and Adenovirus brought key insights into the structure and function of the Retinoblastoma protein (Rb). Knudsen's model of the two-hit hypothesis to explain patterns of hereditary and sporadic retinoblastoma provided the foundation for the tumor suppressor hypothesis that ultimately led to the cloning of the Rb gene. The discovery that SV40 and Adenovirus could cause tumors when inoculated into animals was startling not only because SV40 had contaminated the poliovirus vaccine and Adenovirus was a common cause of viral induced pneumonia but also because they provided an opportunity to study the genetics and biochemistry of cancer. Studies of mutant forms of these viruses led to the identification of the E1A and Large T antigen (LT) oncogenes and their small transforming elements including the Adenovirus Conserved Regions (CR), the SV40 J domain and the LxCxE motif. The immunoprecipitation studies that initially revealed the size and ultimately the identity of cellular proteins that could bind to these transforming elements were enabled by the widespread development of highly specific monoclonal antibodies against E1A and LT. The identification of Rb as an E1A and LT interacting protein quickly led to the cloning of p107, p130, p300, CBP, p400 and TRRAP and the concept that viral transformation was due, at least in part, to the perturbation of the function of normal cellular proteins. In addition, studies on the ability of E1A to transactivate the Adenovirus E2 promoter led to the cloning of the heterodimeric E2F and DP transcription factor and recognition that Rb repressed transcription of cellular genes required for cell cycle entry and progression. More recent studies have revealed how E1A and LT combine the activity of Rb and the other cellular associated proteins to perturb expression of many genes during viral infection and tumor formation.

The story of SV40-induced tumorigenesis and cellular transformation is intimately entwined with the development of modern molecular biology. Because SV40 and other viruses have small genomes and are relatively easy to manipulate in the laboratory, they offered tractable systems for molecular analysis. Thus, many of the early efforts to understand how eukaryotes replicate their DNA, regulate expression of their genes, and translate mRNA were focused on viral systems. The discovery that SV40 induces tumors in certain laboratory animals and transforms many types of cultured cells offered the first opportunity to explore the molecular basis for cancer. The goal of this article is to highlight some of the experiments that have led to our current view of SV40-induced transformation and to provide some context as to how they contributed to basic research in molecular biology and to our understanding of cancer.

Middle T antigen (MT) is the principal oncogene of murine polyomavirus. Its study has led to the discovery of the roles of tyrosine kinase and phosphoinositide 3-kinase (PI3K) signaling in mammalian growth control and transformation. MT is necessary for viral transformation in tissue culture cells and tumorigenesis in animals. When expressed alone as a transgene, MT causes tumors in a wide variety of tissues. It has no known catalytic activity, but rather acts by assembling cellular signal transduction molecules. Protein phosphatase 2A, protein tyrosine kinases of the src family, PI3K, phospholipase Cgamma1 as well as the Shc/Grb2 adaptors are all assembled on MT. Their activation sets off a series of signaling cascades. Analyses of virus mutants as well as transgenic animals have demonstrated that the effects of a given signal depend not only tissue type, but on the genetic background of the host animal. There remain many opportunities as we seek a full molecular understanding of MT and apply some of its lessons to human cancer.

Objective: Serous borderline ovarian tumors (SBOT) are slow growing, noninvasive ovarian epithelial neoplasms, which tend to recur as low-grade invasive carcinomas (LGC) with a much worse prognosis. We investigated the molecular basis of this progression., Methods: We established cultures of three SBOTs and one LGC from tumor biopsies, and inactivated p53, Rb and PP2A in the cells with SV40 large T (LT) and small T (ST) antigen. They were examined for cadherins by immunofluorescence and immunoblotting, invasiveness in Boyden chambers, motility by scratch-wound healing assay, anchorage independence by growth in agarose, and protease activity by gelatin zymography, immunoassay and colorimetry. Cells were overexpressed with N-cadherin using an adenovirus., Results: Inactivation of p53, Rb and PP2A by SV40 LT/ST antigen resulted in greatly enhanced growth potential, invasiveness, motility and anchorage independence, and in epithelio-mesenchymal transition, as indicated by morphology and substitution of N-cadherin for E-cadherin. Overexpressed N-cadherin did not induce invasiveness of SBOT cells and there was no consistent change in protease activities, suggesting that these were not primary effectors of the enhanced neoplastic characteristics. Low passage LGC cells were more invasive than SBOT cells, but this difference disappeared with the introduction of LT/ST into the two cell types., Conclusion: Downregulation or inactivation of p53, Rb and/or PP2A plays a role in the progression from SBOT to invasive ovarian carcinomas.

Polyomaviral vectors are generated by transfecting 293T cells with three sets of DNAs: DNA for the expression of simian virus 40 (SV40) T antigen; DNA for the expression of SV40 capsid proteins, and vector DNA harboring a reporter gene expression cassette carrying a SV40 origin. The vector DNA harbors a minimal sequence originating from SV40, and thus can carry a longer transgene. Moreover, the viable recombinants are not detectable in the vector preparation, and the vectors can transduce the DNA with efficiency similar to that of virions. Vector particles bearing capsid proteins of BK virus, JC virus, and B-lymphotropic papovavirus instead of SV40 were prepared, and they exhibited differential efficiency of gene transduction to the target cells. This method can be used to develop a surrogate system to study the functions of capsid proteins of polyomaviruses and to generate a set of polyomaviral vectors targeted at specific cell types.

Viral DNA is maintained episomally in SV40 infected mesothelial cells and virus is produced at low but steady rates. High copy numbers of the viral DNA are maintained in a WT infection where both early antigens are expressed. In the absence of ST, cells are immortal but non-transformed and the infected cells maintain only a few copies of episomal viral DNA. We show that ST expression is necessary for the maintenance of high copy numbers of viral DNA and that the PP2A binding ability of ST plays a role in genome maintenance. Interestingly, an siRNA to the virus late region downregulates virus copy number and virus production but does not prevent the anchorage-independent growth of these cells. Furthermore, addition of virus neutralizing antibody to culture media also decreases copy numbers of viral DNA in WT-infected cells, suggesting that virus production and re-infection of cells may play a role in maintaining the persistent infection.

To better understand the relationship between epitope variation and tumor escape from immune surveillance, SV40 T antigen-transformed B6/K-0 cells were subjected to selection with individual CTL clones specific for the SV40 T antigen H-2D(b)-restricted epitopes I or V. CTL-resistant populations were isolated from a majority of the selection cultures and substituted epitope sequences were identified within most of the resistant populations. Tag sequences deleted of all or portions of the selection-targeted epitope were identified, but in lower numbers compared to epitope sequences bearing single residue substitutions. Relatively few flanking residue substitutions were identified, and only in epitope I-targeted selections. The diversity (numbers and epitope residue locations) of substituted epitope residue positions varied between selections. These findings suggest that the scope of spontaneously occurring mutations that could allow for escape from individual CD8+ T cell clones is large.

The host range activity of SV40 has been described as the inability of mutant viruses with deletions in the C terminal region of large T Ag to replicate in certain types of African green monkey kidney cells. We constructed new mutant viruses expressing truncated T Ag proteins and found that these mutant viruses exhibited the host range phenotype. The host range phenotype was independent of acetylation of T Ag at lysine 697. Co-expression of the C terminal domain of T Ag (aa 627-708) in trans increased both T Ag and VP1 mRNA as well as protein levels for host range mutant viruses in the restrictive cell type. In addition, the T Ag 627-708 fragment promoted the productive lytic infection of host range mutant viruses in the nonpermissive cell type. The carboxyl-terminal region of T Ag contains a biological function essential for the SV40 viral life cycle.

Simian virus 40 (SV40) is associated with some systemic non-Hodgkin's lymphomas (NHL) among HIV-positive patients, based on assays for viral DNA sequences. To investigate the possible production of the viral transforming protein, we examined age-matched case-control specimens from patients with HIV/AIDS for the expression of SV40 large tumor antigen (T-ag). Masked specimens initially examined by polymerase chain reaction (PCR) for polyomavirus and herpesvirus DNA sequences were assessed for the expression of SV40 T-ag and phenotypic lymphocyte markers by immunohistochemistry (IHC). Fifty-five systemic NHL and 25 nonmalignant lymphoid and malignant nonlymphoid tissue control cases from two HIV community programs in Texas and New Jersey were scored for IHC positivity without knowledge of the PCR results. IHC showed expression of SV40 T-ag among B-cell lymphomas, whereas none of the control tissue samples were positive for T-ag (12/55, 22% vs. 0/25, 0%; P = 0.01). SV40 T-ag expression was detected only in B-cell lymphoma specimens that contained SV40 DNA sequences. Not all lymphoma cells in a positive specimen stained for T-ag, and the reaction was lower intensity than observed in SV40 hamster tumors. SV40 T-ag was demonstrated in both primary and recurrent tumors from one patient. A germinal center B-cell-like (GCB) profile was more frequently expressed by SV40-positive tumors than in Epstein-Barr virus (EBV)-related lymphomas (10/12, 83% vs. 6/13, 46%; P = 0.05), whereas a non-GCB phenotype was more frequent in EBV-positive than in SV40-positive lymphomas (7/13, 54% vs. 2/12, 17%; P = 0.05). This study shows that SV40 gene expression occurs in a fraction of cells in some B-cell lymphomas among patients with HIV/AIDS.

Background. - We have established proliferating human cardiomyocyte cell lines derived from non-proliferating primary cultures of adult ventricular heart tissue, using a novel method that may be applicable to many post-mitotic primary cultures. Methods and results. - Primary cells from human ventricular tissue, were fused with SV40 transformed, uridine auxotroph human fibroblasts, devoid of mitochondrial DNA. This was followed by selection in uridine-free medium to eliminate unfused fibroblasts. The fused cells were subcloned and screened for cell type-specific markers. Four clones (AC1, AC10, AC12, AC16) that express markers characteristic of cardiomyocytes were studied. Clones were homogeneous morphologically, and expressed transcription factors (GATA4, MYCD, NFATc4), contractile proteins such as alpha- and beta-myosin heavy chain, alpha-cardiac actin, troponin I, desmoplakin, alpha actinin, the muscle-specific intermediate filament protein, desmin, the cardiomyocyte-specific peptide hormones, BNP, the L-type calcium channel alpha1C subunit and gap junction proteins, connexin-43 and connexin-40. Furthermore, dye-coupling studies confirmed the presence of functional gap junctions. EM ultra structural analysis revealed the presence of myofibrils in the subsarcolemmal region, indicating a precontractile developmental stage. When grown in mitogen-depleted medium, the AC cells stopped proliferating and formed a multinucleated syncytium. When the SV40 oncogene was silenced using the RNAi technique, AC16 cells switched from a proliferating to a more differentiated quiescent state, with the formation of multinucleated syncyntium. Concurrently, the cells expressed BMP2, an important signaling molecule for induction of cardiac-specific markers, that was not expressed by the proliferating cells. The presence of the combination of transcription factors in addition to muscle-specific markers is a good indication for the presence of a cardiac transcription program in these cells. CONCLUSIONS. - Based on the expression of myogenic markers and a fully functional respiratory chain, the AC cells have retained the nuclear DNA and the mitochondrial DNA of the primary cardiomyocytes. They can be frozen and thawed repeatedly and can differentiate when grown in mitogen-free medium. These cell lines are potentially useful in vitro models to study developmental regulation of cardiomyocytes in normal and pathological states.

Transformation of human cells requires both SV40 large T and small t antigens. Plasmids that contained mutations in the amino-terminal dnaJ domain of the early region fail to transform human diploid fibroblasts. However, large T dnaJ mutants can be rescued by plasmids that express early region products other than large T antigen. The protein found to be responsible for such complementation was the third early region product, 17KT. Similar to large T, this protein reduces levels of the retinoblastoma-related protein, p130, and stimulates cell-cycle progression of quiescent fibroblasts, two activities of large T that are disrupted by dnaJ mutations.

Epithelial ovarian cancer is the most common form of gynaecological malignancy. This lethal disease is thought to arise in ovarian surface epithelial (OSE) cells. The biology of these cells is not well understood, due to the limited amount of tissue that can be obtained from a single biopsy and their limited life span in culture. To overcome these problems, we have conditionally immortalised OSE cells with the catalytic subunit of telomerase (hTERT) and a temperature-sensitive form of SV40 Large T antigen (tsT). We have maintained these cells (designated OSE-C2) in culture for more than 100 population doublings after introduction of the immortalising genes. Early passage OSE-C2 cells have a near-tetraploid karyotype and exhibit a dual mesenchymal-epithelial phenotype, with consistent expression of vimentin and variable expression of cytokeratins and type III collagen, and absence of E cadherin expression. OSE-C2 cells proliferate steadily at the permissive temperature of 33 degrees C, but fail to increase in number at the nonpermissive temperature of 39 degrees C. Serum-deprived OSE-C2 cells are stimulated to grow at 33 degrees C by EGF, whereas they are growth inhibited at 33 degrees C by TGFbeta in the presence or the absence of serum. When temperature shifted to the nonpermissive temperature, OSE-C2 cells modulate to a more mesenchymal phenotype, and a proportion of the cells undergo senescence and/or apoptosis. Moreover, at the nonpermissive temperature, the levels of p53 and SV40 Large T antigen diminish, whilst the level of p21 increases, whereas the level of p16 and telomerase activity is unchanged. This experimental system shows that expression of telomerase alone only allows limited proliferative potential of OSE cells; expression of tsT is necessary to maintain these cells in culture for longer periods, perhaps by its ability to inactivate components of the p53/Rb pathway. OSE-C2 cells may be useful in studying the physiology and differentiation of human OSE cells and provide insight into the poorly understood earliest stages of epithelial ovarian cancer.

Development of the musculoskeletal system requires coordinated formation of distinct types of tissues, including bone, cartilage, muscle, and tendon. Compared to muscle, cartilage, and bone, cellular and molecular bases of tendon development have not been well understood due to the lack of tendon cell lines. The purpose of this study was to establish and characterize tendon cell lines. Three clonal tendon cell lines (TT-E4, TT-G11, and TT-D6) were established using transgenic mice harboring a temperature-sensitive mutant of SV40 large T antigen. Proliferation of these cells was significantly enhanced by treatment with bFGF and TGF-beta but not BMP2. Tendon phenotype-related genes such as those encoding scleraxis, Six1, EphA4, COMP, and type I collagen were expressed in these tendon cell clones. In addition to tendon phenotype-related genes, expression of osteopontin and Cbfal was observed. These clonal cell lines formed hard fibrous connective tissue when implanted onto chorioallantoic membrane in ovo. Furthermore, these cells also formed tendon-like tissues when they were implanted into defects made in patella tendon in mice. As these tendon cell lines also produced fibrocartilaginous tissues in tendon defect implantation experiments, mesenchymal stem cell properties were examined. Interestingly, these cells expressed genes related to osteogenic, chondrogenic, and adipogenic lineages at low levels when examined by RT-PCR. TT-G11 and TT-E4 cells differentiated into either osteoblasts or adipocytes, respectively, when they were cultured in cognate differentiation medium. These observations indicated that the established tendon cell line possesses mesenchymal stem cell-like properties, suggesting the existence of mesenchymal stem cell in tendon tissue.

The ectopic expression of antibody fragments within mammalian cells is a challenging approach for interfering with or even blocking the biological function of the intracellular target. For this purpose, single-chain Fv (scFv) fragments are generally preferred. Here, by transfecting several mammalian cell lines, we compared the intracellular behavior of two scFvs (13R4 and 1F4) that strongly differ in their requirement of disulphide bonding for the formation of active molecules in bacteria. The scFv 13R4, which is correctly folded in the bacterial cytoplasm, was solubly expressed in all cell lines tested and was distributed in their cytoplasm and nucleus, as well. In addition, by appending to the 13R4 molecules the SV40 T-antigen nuclear localisation signal (NLS) tag, cytoplasmic-coexpressed antigen was efficiently retargeted to the nucleus. Compared to the scFv 13R4, the scFv 1F4, which needs to be secreted in bacteria for activity, accumulated, even with the NLS tag, as insoluble aggregates within the cytoplasm of the transfected cells, thereby severely disturbing fundamental functions of cell physiology. Furthermore, by replacing the NLS tag with a leucine-rich nuclear export signal (NES), the scFv 13R4 was exclusively located in the cytoplasm, whereas the similarly modified scFv 1F4 still promoted cell death. Coexpression of NES-tagged 13R4 fragments with nuclear antigen promoted its efficient retargeting to the cytoplasm. This dominant effect of the NES tag was also observed after exchange of the nuclear signals between the scFv 13R4 and its antigen. Taken together, the results indicate that scFvs that are active in the cytoplasm of bacteria may behave similarly in mammalian cells and that the requirement of their conserved disulphide bridges for activity is a limiting factor for mediating the nuclear import/export of target in a mammalian cell context. The described shuttling effect of antigen conferred by a soluble scFv may represent the basis of a reliable in vivo assay of effective protein- protein interactions.

The polyomavirus hr-t class of mutants has served as a major prototype to study the function of middle T + small T in the virus lytic cycle, Biochem. Biophys. Acta 695 (2), 69-95). The properties of these middle T + small T defective mutants were defined by comparisons with "wild-type" strains reconstructed by marker rescue. Similar comparisons in the A2 genetic background have revealed a number of differences, J. Virol. 75, 8380-8389). Here we describe a major divergence in their effects on cell-cycle progression of both permissive mouse NIH3T3 cells and semipermissive Fischer rat FR3T3 cells. Infection of NIH3T3 or FR3T3 cells in serum-rich medium with wild-type A2 (WTA2) or WTA2-derived middle T + small T-defective mutants did not perturb cell cycling, tested up to entry into the third cycle. In contrast, infection with four hr-t mutants analyzed, examined in detail with mutant B2, resulted in an accumulation of cells in G2/M in a dose-dependent and serum-independent manner. The arrest began in the first cell cycle. At multiplicities of infection above 10 PFU/cell, 50-80% of the cell population became arrested by the end of the second cycle. FR3T3 arrested cells detached from the monolayer with a rounded up morphology. Three other hr-t mutants investigated were also found to arrest cells in G2/M. Expression of middle T and/or small T either in trans or in cis did not abrogate this cell-cycle arrest, as demonstrated in the latter case with the middle T + small T expressing strain "wtB2" obtained by repair of the B2 deletion. In FR3T3 cells, the induction of a cell-cycle arrest by wtB2 was accompanied by a severe delay and reduction in neoplastic transformation relative to WTA2 used at equal dose. Mutation(s) in the C-terminal domain of large T antigen, upstream of the site-specific DNA binding activity, is necessary for the cell-cycle block. The possible causes for the cell-cycle block are discussed.

Primary rabbit kidney proximal tubule (RPT) cells (S.D. Chung et al., 1982, J. Cell Biol. 95, 118-126) were transfected with the vector pRSV-T, which contains SV40 early region genes. After the third passage (when normal cells had stopped dividing), individual colonies formed in cultures transfected with pRSV-T. Clonal isolates (RPT-I cells) could be obtained in a simple and reproducible manner. Southern analysis of clone RPT-I8 indicated the presence of SV40 early region genes. Nuclear SV40 T was detected. After 23 passages, and subcloning, RPT-I8 (and subclones) was found to express renal proximal tubule markers to a similar extent, indicating that the phenotype was stable. Nevertheless, the activities of the Na(+)/glucose cotransport system, gamma-glutamyl transpeptidase and alkaline phosphatase, were reduced as compared with primary cultures. Western analysis indicated that the level of Na(+)/glucose cotransporters was maintained in RPT-I8 cells, when compared with intact proximal tubules and primary cultures. Thus, the reduction in alpha-MG uptake in RPT-I8 cells may be attributed to other types of cellular alterations, including changes in energy metabolism. Indeed, growth in glucose-free medium was not observed in RPT-I8 cell cultures, suggesting that unlike primary RPT cells (J. C. Chung et al., 1992, J. Cell. Physiol. 150, 243-250), the gluconeogenic pathway was not intact.

The availability of cell lines that retain their differentiation programs is important for the study of differentiated cell types and the development of cell therapies. DNA tumor virus genes are often used to establish cell lines from primary culture for the analysis of cell-specific functions. To ascertain whether viral immortalizing or transforming genes differed in their effects on cellular differentiation programs, the E1A 12S (WT12S) gene of adenovirus and the large T antigen (LT) gene of SV40 were used to derive stable cell lines from primary kidney. The resultant cell types exhibited very different morphologies, growth and behavior patterns, differentiation states, and plasticities. Renal cells immortalized by LT exhibited branching tubulogenesis in response to Matrigel. This was in contrast to their behavior under normal culture conditions, wherein they were less differentiated, very nonadhesive, very rapidly growing, and transformed. These cells coexpressed adult epithelial (keratin) and embryonic mesenchymal (vimentin, osteopontin, FSP1, PAX-2, and WT1) genes. WT12S-immortalized cells grown on or in Matrigel formed cysts or tubules, consistent with their expression profiles, which consisted of both epithelial and adult kidney markers (E-cadherin, alpha-catenin, circumferential actin filaments (CAF), alkaline phosphatase, aminopeptidase M, BMP7, or podocalyxin), but not embryonic/mesenchymal markers (PAX-2 or WT1). The WT12S-expressing cells were well differentiated, adhesive, slow growing, and nontransformed. Thus, cells expressing WT12S maintained their original differentiation status and were less sensitive to reprogramming, while cells expressing LT were dedifferentiated, but had the potential for reprogramming by exogenous factors.

Central nervous system progenitor cells that are self-renewing in culture and also differentiate under controlled conditions are potentially useful for developmental studies and for cell-based therapies. We characterized growth and plasticity properties and gene expression in a rat mesencephalic cell line, AF5, that was immortalized with an N-terminal fragment of SV40 large T (T155g). For over 150 population doublings in culture, the growth rate of AF5 cells remained steady, the cells remained responsive to bFGF, and telomerase activity and telomere lengths were unchanged. While karyotype analyses revealed some chromosomal abnormalities, these were also unchanged over time; additionally, no mutations in p53 gene sequences were found, and wild-type p53 activation was normal. AF5 cells produced PDGF, TGFbeta1, TGFbeta2, and bFGF. Similar to primary progenitor cells, AF5 cells retained their plasticity in culture; they could be propagated in an undifferentiated state as "neurospheres" in serum-free media or as adherent cultures in serum-containing media, and they differentiated when allowed to become confluent. Adherent subconfluent actively growing cultures expressed a marker for immature neurons, nestin, while few cells expressed the mature neuronal cell marker betaIII-tubulin. Confluent cultures ceased growing, developed differentiated morphologies, contained few or no nestin-expressing cells, and acquired betaIII-tubulin expression. Global gene expression was examined using a 15,000 gene microarray, comparing exponential growth with and without bFGF stimulation, and the differentiated state. The AF5 cell line exhibited stable genetic and growth properties over extended periods of time, while retaining the ability to differentiate in vitro. These data suggest that the AF5 cell line may be useful as an in vitro model system for studies of neural differentiation., ((c) 2002 Elsevier Science (USA).)

Defining the ability of simian virus 40 (SV40) to transform human cells has become of even greater importance with the increased understanding that this virus may play a role in some human malignancies. This report documents the requirement for viral small-t (ST) antigen in large-T (LT)-driven transformation of primary fibroblasts, a requirement that cannot be met by a well-known oncogene, c-Ha-ras (EJ-ras), which can cooperate with LT in rodent systems. The cellular gene telomerase is not essential for transformation, although transformed clones are not immortal without it. Similarly, an immortal mesothelial cell line has been developed using LT and telomerase. Immortalized mesothelial cells are morphologically normal, but can be transformed by introduction of ST, or ST + ras, but not by ras alone. It is likely that ST will be required along with LT for transformation of most human cell types.

Targeted expression of the SV40 large T-antigen oncoprotein (T-Ag) induces cardiomyocyte proliferation in the atria and ventricles of transgenic mice. Previous studies have identified the p53 tumor suppressor, p107 (a homologue of the retinoblastoma tumor suppressor), and p193 (a novel BH3 only proapoptosis protein) as prominent TAg binding proteins in cardiomyocyte cell lines derived from these transgenic mice. To further explore the significance of these protein-protein interactions in the regulation of cardiomyocyte proliferation, a transgene comprising the human atrial natriuretic factor (ANF) promoter and sequences encoding a mutant T-Ag lacking the p53 binding domain was generated. Repeated micro-injection of this DNA gave rise to genetically mosaic animals with minimal transgene content, suggesting that widespread cardiac expression of mutant T-Ag was deleterious. This notion was supported by the observation that the transgene was selectively lost from the cardiac myocytes (but not the cardiac fibroblasts) in the mosaic animals. Crosses between the mosaic mice and animals expressing a cardiac restricted dominant negative p53 resulted in transgene transmission with ensuing overt cardiac tumorigenesis. Transfection of the mutant T-Ag in embryonic stem (ES) cell-derived cardiomyocytes resulted in wide-spread cell death with characteristics typical of apoptosis. Co-transfection with a dominant negative p53 transgene rescued mutant TAg-induced cell death in the ES-derived cardiomyocyte cultures, resulting in a marked proliferative response similar to that seen in vivo with the rescued transgenic mouse study. These results indicate that T-Ag expression in the absence of p53 functional abrogation results in cardiomyocyte death., (Copyright 2001 Academic Press.)

Objective: The aim of this study was to study the pattern of protein kinase expression in a culture model of epithelial ovarian carcinogenesis., Methods: Cultures of normal human ovarian surface epithelium (OSE), OSE from women with BRCA1 mutations, a cell culture model of preneoplastic (SV40 T-antigen-immortalized, nontumorigenic) and neoplastic (SV40-E-cadherin transfected, tumorigenic) OSE, and three ovarian cancer cell lines were used to represent OSE phenotypes of different genetic backgrounds and at different, progressive stages of neoplastic transformation. The protein kinase network signaling was studied by Western blotting, simultaneously using multiple antibodies for specific protein kinases., Results: High levels of cGMP-dependent protein kinase were found in normal and preneoplastic OSE, but were absent in neoplastic OSE. In contrast, expression of MEK6 was detected exclusively in neoplastic OSE. The expressions of casein kinase II (CK2), p38 mitogen-activated protein kinase (MAPK), cyclin-dependent kinase, and the phosphatidylinositol 3-kinase (PI3K) effectors Akt2 and p70 S6 kinase (S6K) were several-fold higher in neoplastic OSE than in normal OSE, whereas the expressions of the MAPKs extracellular signal-regulated kinases ERK1 and -2 were unchanged. Importantly, constitutive phosphorylation of Akt2 and p70 S6K, as found in neoplastic OSE, was also observed in overtly normal OSE from women with predisposing BRCA1 gene mutations., Conclusions: These data demonstrate that different repertoires of downstream signaling proteins, particularly those of the MEK6-p38 MAPK-CK2 pathway and the PI3K pathway, are correlated with phenotypic manifestations of a cell culture model of OSE at progressive stages in the development of ovarian cancer. Changes in PI3K effectors are already found in overtly normal OSE from women with BRCA1 mutations., (Copyright 2001 Academic Press.)

The SV40 large T-antigen (TAg) has proven useful in studying pathways involved with cell division and tissue homeostasis. TAg disrupts the normal action of tumor suppressors pRb and p53. It is unclear whether T-antigen inhibition of p53 and pRb is sufficient for oncogenic transformation or if additional T-antigen activities are required. To pursue this question, cell lines were generated that coexpress an amino-terminal fragment of T-antigen (TAgN136), which has been shown to be sufficient to block pRb function, together with a dominant-negative p53. Neither focus formation nor saturation density was enhanced by coexpression of the dominant-negative p53 molecule, p53DD, along with TAgN136. Furthermore, a full-length TAg mutant incapable of binding p53 was capable of relieving contact inhibition, a hallmark of transformation. These results suggest the presence of a novel transforming activity in addition to the binding and inactivation of p53, requiring TAg amino acids 137 to 708., (Copyright 2001 Academic Press.)

Under standard culture conditions, normal human mammary epithelial cells (HMECs) divide a limited number of times before proliferation ceases in a growth-arrested state referred to as selection. Cells that have undergone spontaneous loss of p16(INK4a) expression due to hypermethylation of the p16(INK4a) CpG island emerge from selection and proliferate for an extended, but limited, period before senescence. Here we show, as expected, that selection was bypassed by expression of SV40 large T-antigen proteins containing an intact pRb-binding domain in preselection cells. These cells were immortalized with high efficiency (seven of nine separate cultures). Also as expected, postselection cells were immortalized by expression of the human papillomavirus-16 E6 oncoprotein (four of four cultures), which inactivates p53 protein. In contrast, we found that expression of SV40 large T-antigen protein, which also inactivates p53, was poorly maintained in postselection cultures due to its growth-suppressive effects; consequently, these cells became immortalized at low efficiency (one of 11 cultures). Reexpression of p16(INK4a) in postselection HMECs by the demethylating agent, 5-azacytidine, or transfection of a p16(INK4a) expression plasmid did not restore the ability of these cells to undergo SV40-induced transformation. Postselection HMECs are a widely used in vitro model system, but these observations indicate they have undergone changes in gene expression in addition to loss of p16(INK4a) expression., (Copyright 2001 Academic Press.)

Objectives: Epithelial ovarian cancers are considered to arise from neoplastic transformation of the ovarian surface epithelium (OSE). However, the earliest events in ovarian carcinogenesis have not been clearly defined because patients are often diagnosed in the advanced stages and useful in vivo and in vitro experimental systems using human OSE cells are lacking. We aimed to improve the availability of experimental models for the study of human ovarian carcinogenesis., Methods: Subcultured human OSE cells were transfected with SV40 large T antigen. Resulting OSE cell lines were characterized using immunocytochemistry and tested tumorigenicity., Results: Six immortalized OSE cell lines were obtained. All cell lines essentially retained the original morphological features of normal OSE cells and showed higher proliferation rates and saturation density. Although they were all nontumorigenic in athymic mice, OSE2b-2 sv cells, which were selected in soft agar from colonies of an SV40 large T antigen-expressing transfectant, OSE2b sv, produced tumors on the peritoneal surface, mesothelium, and diaphragm and induced ascites after being injected intraperitoneally. Solid tumors also grew when mice were inoculated subcutaneously. The tumor cells were formed in a solid sheet arrangement and no evidence of glandular or squamous differentiation was present. They were weakly immunostained with an antibody against cytokeratin, and intercellular junctions resembling attachment devices were ultrastructurally present between cells. The tumors were histologically diagnosed as undifferentiated carcinomas., Conclusions: The established cell lines may provide a model system to investigate the mechanisms of cytogenic and molecular changes from normal OSE cells through the various steps of transformation., (Copyright 2001 Academic Press.)

Although nearly 60% of mesotheliomas contain SV40 early region DNA sequences, the role of T/t antigens in initiating and maintaining the transformed state of mesothelioma cells remains unclear. The majority of mesothelioma cells which contain SV40 early region sequences exhibit extremely low basal expression of SV40 oncoproteins; however, T/t antigen expression can be induced under conditions of cellular stress. Abrogation of SV40 T/t expression by antisense techniques induces apoptosis in part via restoration of p53 function, and enhances chemosensitivity in SV40 (+) MPM cells by mechanisms which have not been fully elucidated. This review briefly summarizes our ongoing efforts to define the role of SV40 oncoproteins in modulating the malignant phenotype of mesothelioma cells, and highlights strategies which may prove efficacious in vivo for circumventing SV40 T/t antigen expression in mesotheliomas., (Copyright 2001 Academic Press.)

The regulatory region (RR) of simian virus 40 (SV40) contains enhancer/promoter elements and an origin of DNA replication. Natural SV40 isolates from simian brain or kidney tissues typically have an archetypal RR arrangement with a single 72-basepair enhancer element. A rare simpler, shorter SV40 RR exists that lacks a duplicated sequence in the G/C-rich region and is termed protoarchetypal. Occasionally, SV40 strain variants arise de novo that have complex RRs, which typically contain sequence reiterations, rearrangements, and/or deletions. These variants replicate faster and to higher titers in tissue culture; we speculate that such faster-growing variants were selected when laboratory strains of SV40 were initially recovered. SV40 strains with archetypal RRs have been found in some human brain tumors. The possible implications of these findings and a brief review of the SV40 RR structure are presented., (Copyright 2001 Academic Press.)

A HeLa cell line expressing the green fluorescent protein fused to the SV40 T-antigen nuclear localization signal (EGFP-NLS) was established. Fluorescence in these cells was confined to the nuclei. After poliovirus infection, cytoplasmic fluorescence in a proportion of cells could be detected by 1 h postinfection (p.i.) and in virtually all of the fluorescent cells by 2 h p.i. The relocation could be prevented by cycloheximide but not by inhibition of poliovirus replication by guanidine. HCl. Nuclear exit of a protein composed of three copies of GFP fused to the NLS also occurred upon poliovirus infection. A similar redistribution of EGFP-NLS took place upon infection with coxsakievirus B3 and, to a lesser extent, with vesicular stomatitis virus. The EGFP-NLS efflux was not due to the loss of NLS. Thus, some positive-strand and negative-strand RNA viruses trigger a rapid nonspecific relocation of nuclear proteins., (Copyright 2000 Academic Press.)

The amino termini of polyomavirus T antigens contain LXCXE and J domains, which are necessary for binding and inactivating the retinoblastoma family of tumor suppressors. Both of these motifs are found in the JC virus (JCV) early proteins T'(135), T'(136), and T'(165), leading to the suggestion that these recently discovered proteins complement the cell-cycle-deregulating function of the JCV large T antigen (TAg). To investigate this hypothesis, the three JCV T' proteins were produced in a baculovirus expression system and purified by immunoaffinity chromatography. To facilitate purification, hybridomas that secrete antibodies recognizing amino-terminal epitopes of JCV early proteins were produced. Potential interactions between the early viral proteins and the cellular proteins pRB, p107, and p130 were investigated by incubating purified JCV TAg and T' proteins with extracts of MOLT-4 cells, a human T cell line. The four viral proteins preferentially bound hypophosphorylated species of the cellular proteins and exhibited the highest binding affinity to p107 and the lowest affinity to pRB. TAg and T'(165) bound more pRB and less p107 than did T'(135) and T'(136); T'(165) also bound less p130 than the other three early proteins. Results of these in vitro interactions were compared to those obtained in vivo using POJ cells, a transformed human glial cell line that expresses JCV early proteins, relatively high levels of pRB and p107, and low levels of p130. Most of the pRB in POJ cells is hyperphosphorylated, and only a fraction of the hypophosphorylated form(s) of pRB is bound by the viral proteins. In contrast, only hypophosphorylated p130 is detected in the transformed cells, and most of this protein was found in complex with the viral proteins. Finally, nearly all of the p107 in POJ cells is bound by the JCV proteins., (Copyright 2000 Academic Press.)

Medulloblastoma, a malignant invasive tumor of the cerebellum, is one of the most common neoplasms of the nervous system in children. Utilization of the human neurotropic virus JC virus (JCV) early gene T-antigen allowed the development of a transgenic animal that models human medulloblastoma. Here we describe the characterization of two distinct populations of cells derived from the JCV-induced mouse medulloblastoma. Results from immunohistochemical and biochemical studies revealed the expression of T-antigen in some but not all tumor cells. In T-antigen-producing cells, T-antigen was found in association with wild-type p53 and pRb, two tumor suppressors that control cell growth and differentiation. In cells that lack expression of T-antigen, a novel mutant p53 with a deletion between residues 35 and 123 was detected. Morphological differences were observed between the two populations of cells, though there was no significant difference in their growth rates. However, subcutaneous transplantation of the T-antigen-positive, but not T-antigen-negative, cells resulted in the development of massive tumors in experimental animals. In light of earlier reports on the association of JCV with human medulloblastoma, the mouse cell lines described in this study may provide a valuable tool for deciphering the pathways involved in the formation and progression of medulloblastoma., (Copyright 2000 Academic Press.)

We have characterized two polyomavirus large T antigen mutants with different properties in viral DNA replication. dl-97, a mutant active in immortalization, exerts a dominant negative effect in viral DNA replication. 13val, which is defective in both immortalization and viral DNA replication, has a lesion in the putative DnaJ domain affecting the block of Rb function., (Copyright 2000 Academic Press.)

Approximately 50% of donor corneas are unsuitable for keratoplasty due to an unacceptably low endothelial cell count. One way of overcoming this problem and minimizing wastage of donor corneas may be to transplant cultured human corneal endothelial cells onto these. In this study, we examined the morphological characteristics and functional attributes of endothelial layers formed after the transplantation of immortalized cells in vitro. Cultured human corneal endothelial cells, immortalized by transfection with a plasmid encoding SV40 T-antigen, were seeded onto human corneas denuded of their own endothelium. Seven days after transplantation the newly established monolayers were examined by light, confocal and scanning electron microscopy. Endothelial pump function was gauged by monitoring changes in corneal thickness during perfusion of the endothelial face. The endothelia formed from transplanted immortalized cells had a cobblestone-like appearance, being composed of polygonal units joined by junctional complexes. The stromal hydration state of corneas bearing such endothelial layers could be controlled during perfusion. This was an active process achieved via the Na(+)/K(+)-ATPase-dependent endothelial pump, as demonstrated by inhibiting the enzyme with ouabain. Transplantation of immortalized human corneal endothelial cells onto recipient corneas led to the establishment of new monolayers which had the morphology of the native ones in organ-cultured corneas. This model provides us with a means of studying the formation and function of corneal endothelial layers in vitro., (Copyright 1999 Academic Press.)

Episomal SV40 (SV40: simian virus 40, Polyomavirus maccacae) has been reported in SV40-transformed human fibroblast cell lines the integrated SV40 sequences of which are unlikely to give rise to episomal copies by recombinational mechanisms. The levels of episomal viral DNA in these lines are high, being easily visualized by ethidium staining of agarose gels after electrophoresis. We find that the episomal mutant gmSV40 in GM637 cells represents a persistent lytic infection that can be cured by treatment with neutralizing antibody, leaving only the chromosomally integrated viral genomes. The finding that maintenance of the gmSV40 in GM637 cells is due to persistent infection raises a note of caution for SV40-transformed lines with episomal SV40 genomes because these lines often are used in studies of DNA replication and repair. An infective center assay that does not depend on plaque formation shows that gmSV40 is a host range mutant, with poor infectivity for CV-1 monkey kidney cells and greatly increased infectivity for human cells. Passage of gmSV40 through monkey kidney cells selects for variants with greatly increased infectivity for monkey cells and, independently, for cytopathic variants that produce plaques. Thus plaque assays can give very unreliable infective center values in studies of host range mutants., (Copyright 1999 Academic Press.)

The infection of monkey kidney (CV-1) cells with simian virus 40 (SV40) stimulates the cells into successive rounds of DNA synthesis without an intervening mitosis, leading to the acquisition of a >G2 DNA content. To elucidate the role of small t antigen in cell cycle progression and in viral replication during infection, studies were performed using an SV40 mutant (dl888) that lacks the ability to produce small t. Initially dl888-infected cells move through the first S phase at roughly the same rate as wild-type infected cells. Upon reaching G2, however, the dl888-infected cells progressed to >G2 at a reduced rate relative to wild-type. The slower rate of entry into >G2 of dl888-infected cells is associated with a decrease in total pRb and an increase in the ratio of hypophosphorylated to hyperphosphorylated pRb. The expression of cyclin D1 and p27(kip1) were elevated in dl888-infected cells compared to wild-type-infected CV-1 cells. Taken together, these results indicate that small t antigen plays a role in stimulating entry into >G2 in SV40-infected CV-1 cells, possibly by affecting the regulation of key cell cycle proteins., (Copyright 1999 Academic Press.)

The tsA58 allele of SV40 large T antigen has the ability to immortalize cells, which is thought to be due, in part, to binding of p53 protein by T antigen at 33 degrees C. At the nonpermissive temperature (39.5 degrees C), it is thought that p53 is released, inducing growth arrest, vulnerability to apoptosis, and loss of the immortal phenotype. In cell lines derived from the rat neostriatum immortalized with tsA58, the toxic agents Adriamycin, cytosine arabinoside, and glutamate induced apoptosis and increased p53 activity and differentiation. The apoptosis and p53-inducing effects of the drugs were not greater at 39.5 degrees C compared to 33 degrees C, suggesting that p53 is not effectively blocked even at 33 degrees C. Growth arrest was not induced under most treatment conditions despite p53 induction. On the other hand, process extension was enhanced at 39.5 degrees C compared to 33 degrees C. Therefore, these cell lines are temperature sensitive with respect to differentiation, but not growth regulation or apoptosis.

Immortalization of chondrocytes by SV40 T Ag has often been reported to trigger the loss of expression of type II collagen, one of the main differentiation markers, although some immortalized chondrocyte lines maintaining a differentiated phenotype have also been described. Here, we show using transient cotransfections in differentiated chondrocytes that, in contrast to c-src, neither SV40 T Ag, nor c-myc, decreases col2a1 transcriptional activity. Then, we report the possibility of immortalizing rabbit articular chondrocytes by expression of SV40 T Ag controlled by the col2a1 promoter and enhancer (pCol2SV). This strategy allows one to select within a population of differentiated chondrocytes those which are able to maintain functional regulation of the col2a1 gene through long-term culture. In precrisis pCol2SV-transfected chondrocytes, all-trans-retinoic acid, a down-regulator of col2a1 expression, induced apoptosis, strongly suggesting the strict control of T Ag expression by col2a1 regulatory sequences. Some pCol2SV-transfected chondrocytes were definitively immortalized, after a short crisis period. However, type II collagen synthesis was restricted to a small proportion of cells, which went on to decrease with subculture, while the proportion of cells expressing T Ag was not affected. In these postcrisis cells, T Ag remained at least partially under the control of functional col2a1 regulatory elements as assessed by all-trans-retinoic acid down-regulation., (Copyright 1999 Academic Press.)

Corneal endothelial cells have a limited capacity for proliferation. Upon transformation with the SV40 large T antigen, however, these cells undergo division and grow rapidly. In order to gain insight into the control mechanisms that determine this proliferative switch, we investigated the expression level and activity of various known cell cycle-regulatory proteins in these cells. Primary human and rabbit corneal endothelial cells were transduced in vitro with a replication-defective adenovirus containing SV40 large T antigen, and subsequently the expression and activity of cell cycle-regulatory proteins was analyzed. Cells transduced with large T antigen exhibited strongly increased activity of cyclin-dependent kinases. This increase correlated with the elevated expression of various cyclin-dependent kinase subunits, such as cyclin A, and to a lesser extent, cyclin D, cdk2, and cdk4. Furthermore, the expression of two cyclin-dependent kinase inhibitors, p21(WAF1) and p27(KIP1), which was high in primary human cells (but not in primary rabbit cells), was strongly reduced in large T-antigen transduced cells. Thus, the remarkably low proliferative activity of normal human corneal endothelial cells appears to be regulated at two levels: the expression of certain cell cycle-regulatory proteins that are essential for cell cycle progression is extremely low (cyclin A) or somewhat low (cdk2 and cdk4); but the amount of p21 and p27, inhibitors of cell cycle progression, is very high. As a consequence, the enzymatic activity of cyclin-dependent kinase is below detectable levels. However, the growth-inhibitory status of these components is clearly reversible: upon transduction with large T antigen, the expression of cyclin A, cyclin D, cdk2, and cdk4 is induced, whereas the expression of p21 and p27 is inhibited, and the cells proliferate. Thus, our study provides insight into the molecular basis of the attenuated proliferation of corneal endothelial cells and suggests potential targets that could be manipulated for the purpose of therapeutic interventions aimed at renewed cell growth., (Copyright 1999 Academic Press.)

We have previously immortalized a mouse submandibular gland (SMG) ductal epithelial cell line, SIMS, from pubertal male mice transgenic for the SV40 large T antigen under the control of the adenovirus 5 E1A promoter. Here we demonstrate the role of the extracellular environment in directing not only the morphogenetic behavior of the cells, but also their functional differentiation in terms of renin expression and secretion. First, we measured renin activity of polarized SIMS cells. Low levels of renin are secreted from both the apical and the basolateral domains; the mechanism appears to be direct as no renin was found to be transcytosed across the cell. Second, we studied homotypic and heterotypic mesenchymal cell interactions with SIMS cells. We found that epithelial-mesenchymal coculture in collagen I gels results in branching tubular morphogenesis of SIMS cells and that significant amounts of renin are secreted, probably into the lumen, as the precursor form, prorenin. Third, we investigated the effects of the basement membrane on SIMS cell morphology and function and found that this structure alone is sufficient to allow expression and secretion of both prorenin and active renin. Finally, we established that SIMS cells can express androgen-regulated genes in a transient transfection assay. In addition, in Matrigel cultures androgen receptor expression appears to be induced, suggesting that the SIMS cell line will be useful for further studies on the molecular basis of the observed high-level expression of SMG-specific genes in male mice., (Copyright 1999 Academic Press.)

Human diploid cells have a limited life span, ending in replicative senescence, in contrast to cell lines derived from tumors, which show an indefinite life span and are immortal, suggesting that replicative senescence is a tumor suppression mechanism. We have utilized introduction of SV40 sequences to develop matched sets of nonimmortal and immortal cell lines to help dissect the mechanism of immortalization and have found that it has multiple facets, involving both SV40-dependent and -independent aspects. These studies have led to the identification of a novel growth suppressor gene (SEN6) as well as providing a model system for the study of cellular aging, apoptosis, and telomere stabilization among other things. It is anticipated that SV40-transformed cells will continue to provide a very useful experimental system leading to insights into the behavior of cells with altered expression of oncogenes and growth suppressor gene products., (Copyright 1998 Academic Press.)

Although primary cultures of human lens epithelial (HLE) cells provide important information concerning the role of epithelium in normal lens and cataract formation, the lack of a cell line precludes a broad range of studies on the metabolism and molecular biology of these cells. We have, therefore developed an HLE cell line. Primary cultures of HLE cells were transfected with plasmid vector DNA containing a large T antigen of SV40. The immortalized cells were characterized with regard to morphology, growth rate, karyotype, and expression of crystallins, aldose reductase and other enzymes. A single clone of the immortalized cells, SRA 01/04, formed a monolayer and grew constantly over 130 passages. Isozyme phenotype showed that SRA 01/04 was of human origin, and the chromosome counts were in the hypotetraploid range. Western blot analysis showed that the cells expressed a very low level of crystallins (alphaA and betaB2) and aldose reductase. Messenger RNA (mRNA) for both alpha and beta crystallins was detected by reverse transcription polymerase chain reaction (RT-PCR) in both early and late passages. Sequence analysis of the PCR products, corresponding to alphaA and betaB2 crystallins in the cell line and in primary cultures of HLE, revealed a 100% match with published human alphaA and betaB2 sequences. These characteristics were unchanged in the cell line in early and late passages. This is the first report of the presence of alphaA and transcripts of mRNA for both alphaA and betaB2 in an established human cell line. This new HLE cell line makes it possible to undertake many future studies on the role of epithelium in lens and cataract formation., (Copyright 1998 Academic Press.)

We have investigated the nuclear transport of the replacement histone H1(0) and have searched for its nuclear localization sequence (NLS). The lysine-rich H1(0) histone differs from the other H1 histones with respect to its mode of expression and to the processing of the respective mRNA. Using the digitonin-permeabilized cell import assay we demonstrate that H1(0) is transported into the nucleus in an energy- and temperature-dependent manner. In competition experiments we show that the transport of H1(0) from the cytoplasm into the nucleus is competed by the SV40 T-antigen-NLS-peptide coupled to HSA, an established substrate of the importin pathway. In transfection studies we have expressed in HeLa cells a series of plasmid constructs containing different fragments of the coding region of the H1(0) histone gene that were fused to the beta-galactosidase gene, and we have determined the subcellular localization of each fusion protein. The results show that H1(0) contains multiple transport-competent sequence elements that can function as NLS and that H1(0) meets the requirements for a transport into the nucleus by an importin-dependent pathway., (Copyright 1998 Academic Press.)

Angiotensin II has been shown to be mitogenic in various cell types. In cultured neonatal cardiomyocytes, we have demonstrated that angiotensin II causes hypertrophy, not hyperplasia. However, fetal or neonatal cardiomyocytes exhibit limited proliferation in primary culture, and are mitotically less potent. In order to determine whether angiotensin II is simply a hypertrophic or hyperplastic growth factor for mitotically-potent cardiomyocytes, we analysed [3H]-thymidine uptake and cell cycle-regulated gene expression using SV40 large T-transformed AT-1 cardiomyocytes. Angiotensin II, alone and in combination with other growth factors, increased [3H]-thymidine uptake in a dose-dependent manner. The mRNA expression of G1 cyclins (Cyclin C, D1, D2, D3) and histone H1-kinase activity by CDK2 increased 6 h after angiotensin II stimulation. Western blot analysis revealed cyclin B1 expression after 18 h , which peaked at 30 h. Histone H1-kinase activity by cdc2 was also increased by angiotensin II, and peaked at 24-36 h, indicating that these changes were cell cycle dependent. Double immunofluorescent photography showed that AT-1 cells incorporated BrdU, and expressed cdc2 by angiotensin II stimulation. [3H]-thymidine and BrdU uptake were blocked by losartan, but not by PD123319. In contrast with neonatal cardiomyocytes, angiotensin II potentiated DNA synthesis and induced cell cycle regulated gene expression in AT-1 cardiomyocytes, and this activity was mediated by the angiotensin II type-1 receptor., (Copyright 1998 Academic Press)

SV40 T antigen downregulates the expression of an important detoxification enzyme, glutathione S-transferase alpha (GSTalpha). We show here that the target of this repression is a 14-bp element common to the human GSTA1 and GSTA2 promoters. This element, which we have named TAGR, is also critical for high-level, constitutive expression from these promoters. The TAGR element does not appear to contain a binding site for any transcription factor known to be present in fibroblasts, although the TAGR element does resemble the binding site for the Ikaros transcription factor found in hematopoietic cells. We also have identified a 47-amino-acid fragment of T antigen that includes amino acids 83-100 and 119-147, which is sufficient to repress transcription from the GSTalpha promoter in transient transcription assays. Thus, GSTalpha repression does not require binding of T antigen to pRb, p300, or p53, since the domains of T antigen required for binding these cellular proteins are missing from this T antigen fragment. We show, however, that this fragment does bind to three cellular proteins with approximate molecular weights of 54, 59, and 94 kDa., (Copyright 1998 Academic Press.)

Transgenic targeting of SV40 large T antigen (Tag) expression to murine cerebellar Purkinje cells induces these normally postmitotic neurons to undergo DNA synthesis and apoptosis. It has been proposed that these effects of Tag are due to the binding of Tag to pRb, which leads to the release and activation of the transcription factor E2F. Here it is reported that E2F and CDC2, the protein product of a gene regulated by E2F, were detectable in the Purkinje cell nuclei of Tag expressing transgenic animals. To directly test whether E2F-1 is part of the mechanism of Tag-induced Purkinje cell degeneration, transgenic mice that overexpress E2F-1 specifically in cerebellar Purkinje cells were generated. Although E2F-1 itself did not affect Purkinje cells, it did accelerate Tag-induced ataxia and Purkinje cell loss, suggesting that E2F-1 can contribute to the mechanism of Tag-induced Purkinje cell degeneration., (Copyright 1998 Academic Press.)

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.