Extended life of human corneal endothelial cells transfected with the SV40 large T antigen

PURPOSE: To transfect human corneal endothelial cells with a plasmid vector coding for the SV40 large T antigen to extend the life of the cells in culture. METHODS: Human corneal endothelial cells were transfected with the SV40 large T antigen-coding plasmid pSV3neo using the electroporation method. Transfected and control cells were propagated in culture until senescence. Polymerase chain reaction and immunofluorescence were used to demonstrate messenger RNA and protein, respectively, for the Simian virus 40 large T antigen in the transfected cells. Polymerase chain reaction and hot blotting were used to demonstrate messenger RNA coding for several growth factors and receptors in transfected and control cells. RESULTS: The transfected cells continued to proliferate to 38 passages (more than 120 population doublings) in culture (control cells, 8 population doublings). Transfected cells, but not control cells, expressed messenger RNA coding for the Simian virus 40 large T antigen. Similarly, immunofluorescent staining with monoclonal antibodies demonstrated that the Simian virus 40 large T antigen protein was present in the nucleus of the transfected cells. Transfected cells were shown to produce messenger RNA coding for epidermal growth factor, epidermal growth factor receptor, basic fibroblast growth factor, fibroblast growth factor receptor-1, interleukin-1 alpha, the interleukin-1 receptor, transforming growth factor beta-1, and the glucocorticoid receptor. Qualitative expression of the messenger RNA coding for each of these modulators was similar in proliferating primary corneal endothelial cells and proliferating or confluent transfected corneal endothelial cells. CONCLUSIONS: In culture, the life of human corneal endothelial cells transfected with a plasmid vector coding for the Simian virus 40 large T antigen is extended. This study suggests that human corneal endothelial cells have the capacity for extensive proliferation, but the proliferation of untransfected cells is regulated through mechanisms that have not yet been characterized.     

Increased expression of nucleoside diphosphate kinases/nm23 in human diploid fibroblasts transformed by SV40 large T antigen or 60Co irradiation

YACHIYODA SZ-5000 is a new model of lithotriptor made in Japan, of which the energy source is microexplosion. Compared with the old type of SZ-1, the water bag is substituted for a hot water bath and ultrasonography as well as fluoroscopy can be used for stone localization. Moreover, this new model is extremely small. The first clinical trial of 32 candidates with urinary tract calculi (34 stones) was performed at our hospital between September 1991 and June 1992. They were 10 women and 22 men between 25 and 71 with a mean of 47.3 years. All patients received no anesthesia. The stone location was: R2 for 16 stones, R3 for 5, U1 for 9, U2 for 1, and U3 for 3. The mean size was 14.1 mm. A mean number of 364, 326 and 324 shock waves were given for the R2, 3, U1, and U2, 3 stones, respectively. The second or third sessions were performed on 7 patients. Obvious symptoms and signs observed during the treatment were; local pain in 9 patients, nausea in 3, hypotension and bradycardia in 6, and hypertension in 3. Posttreatment fever up was found in two patients. In 34 stones, the efficiency evaluated 3 months later was 85.0% as determined by kidney-ureter-bladder X-ray and intravenous pyeography. In conclusion, YACHIYODA SZ-5000 is useful and safe in the management of patients with urinary tract stones.     

Inhibition of proliferation and expression of T-antigen in SV40 large T-antigen gene-induced immortalized cells following transplantations

In this paper the Authors reviewed the recent literature for a more comprehensive and clear vision of the epidemiological and pathological aspects of retroperitoneal sarcomas. The most effective procedures for a an early and accurate diagnosis were identified. Moreover, the different therapeutic choices were taken into account focusing on those provided of the major potential in terms of oncologically valid treatment.     

Negative regulation of the neu promoter by the SV40 large T antigen

We investigated the effect of transforming growth factor-beta 1 (TGF beta) on the proliferation and differentiation of cultured acute promyelocytic leukemia (APL) cells with the chromosomal t(15;17) translocation obtained from four patients to determine the role of TGF beta on growth and differentiation of APL cells. DNA synthesis, determined by 3H-thymidine uptake, was inhibited in the presence and absence of granulocyte colony-stimulating factor (G-CSF) in a dose-dependent manner by TGF beta in APL cells obtained from three of the four cases. TGF beta and G-CSF did not significantly affect the differentiation of APL cells, but all-trans retinoic acid (RA) induced morphological and functional differentiation in all APL cells tested. G-CSF markedly enhanced RA-induced granulocytic differentiation in APL cells obtained from all four cases. In cells in which TGF beta inhibited DNA synthesis, it also inhibited RA-induced granulocytic differentiation of APL cells and, to a greater degree, granulocytic differentiation induced by RA plus G-CSF. These results suggest that TGF beta is a negative regulator of the proliferation and differentiation of APL cells. The significance of TGF beta as an endogenous regulator in differentiation therapy with RA of APL patients is discussed.     

Simian virus 40 large T antigen (SV40LTAg) primer specific DNA amplification in human pleural mesothelioma tissue

BACKGROUND: DNA sequences and immunoreactivity associated with Simian virus 40 transforming factors, large T and small t antigens (SV40LTAg), suggestive of an aetiopathogenetic link have been identified in fresh frozen tissue of a high proportion of recent cases of pleural mesotheliomas from the United States, Italy and Germany. SV40 is not normally infective in man though it can transform human cells in tissue culture. A large cohort of people in the western world was accidentally parenterally inoculated with live SV40 through contaminated polio vaccines given between 1959 and 1961, and this might be a factor in the current continuing rise in the incidence of mesothelioma in the United States, Britain and Europe. The present study investigated the presence of SV40LTAg DNA in recently diagnosed cases of mesothelioma in Britain and the feasibility of detecting the SV40 DNA in archival tissue for retrospective analysis of cases in the peri-vaccination period. METHODS: DNA was extracted from fresh frozen and/or rehydrated formalin fixed, paraffin embedded tissue sections from nine recently diagnosed cases of mesothelioma, nine cases of pulmonary adenocarcinoma, and three reactive pleurae, and amplified by the polymerase chain reaction (PCR) using the primer pairs used previously on fresh frozen tissues-namely, the SV primer set directed at the LTAg gene sequence unique to SV40 and the PYV primer set directed at a sequence shared by SV40 and papovavirus strains BK and JC, respectively. RESULTS: PCR positivity with the SV primer set was restricted to four of the nine cases of mesothelioma. In contrast, six of the nine mesotheliomas, two of the nine adenocarcinomas, and one of the three reactive pleurae showed positivity with the PYV primers. The fresh frozen and corresponding formalin fixed, paraffin embedded tissue results concorded well with each other. CONCLUSIONS: Our data provide evidence for the association of SV40LTAg primer specific DNA with human pulmonary mesothelioma in the British population.     

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.     

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.     

Functional interactions between SV40 T antigen and other replication proteins at the replication fork

The functional interaction of simian virus 40 (SV40) large tumor antigen (T antigen) with DNA polymerase alpha (pol alpha)-primase complex, human single-stranded DNA binding protein (HSSB), and DNA polymerase delta (pol delta) holoenzyme, which includes pol delta, activator I (also called replication factor C), and proliferating cell nuclear antigen, at the replication fork was examined using the purified components that support SV40 DNA replication. Dilution of reaction mixtures during RNA primer synthesis revealed that T antigen remained associated continuously with the fork, while the pol alpha-primase complex dissociated from the complex during oligoribonucleotide synthesis. T antigen unwound duplex DNA from the SV40 core origin at a rate of 200 base pairs/min. Pol alpha-primase complex inhibited the rate of the unwinding reaction, and HSSB, pol alpha, and primase were all required for this effect. These requirements are the same as those essential for DNA primase-catalyzed oligoribonucleotide synthesis (Matsumoto, T., Eki, T., and Hurwitz, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9712-9716). This result suggests that the pol alpha-primase complex interacts with T antigen and HSSB during the unwinding reaction to synthesize RNA primers and that the interaction decreases the rate of T antigen movement. While pol delta holoenzyme can elongate primed DNA chains at a rate of 400-600 nucleotides/min on singly primed phi X174 DNA, the rate of the leading strand synthesis catalyzed by pol delta holoenzyme in the SV40 replication system in vitro was about 200 nucleotides/min. This rate was similar to the unwinding rate catalyzed by T antigen. Thus, the rate of leading strand synthesis catalyzed by pol delta holoenzyme in vitro appears to be limited by the unwinding reaction catalyzed by T antigen.     

Development of thymic carcinoma in transgenic mice expressing SV40 T antigen

The strongest fibrinolytic protease (F-III-2) in the six enzyme proteins purified from earthworm, Lumbricus rubellus [N. Nakajima et al., Biosci. Biotech. Biochem., 57, 1726-1730 (1993)] has been modified chemically with fragmented human serum albumin (mol. wt., 10,000-30,000). The modified enzyme lost the antigenicity of the native enzyme and reacted with the antisera against human serum albumin, the human serum albumin fragments, and the conjugate with the native enzyme to form precipitation lines, which fused with each other. The conjugate was significantly more resistant to inactivation by protease inhibitors in rat plasma. The enzyme was a non-hemorrhagic protein and did not induce platelet aggregation. The enzyme kept potent proteolytic activity for fibrin and fibrinogen than that of human plasmin. The enzyme easily solubilized actual fibrin clots (thrombi) of whole blood induced by thrombin in a rat's vena cava. The continuous fibrinolysis for fibrin suspension in an enzyme reactor system using the modified enzyme immobilized to oxirane-activated acrylic beads has been achieved without any inactivation of the activity at least for more than 1 month. The N-terminal amino acid sequence of the protein was also investigated and the sequence showed local similarity to those of the serine proteases such as plasmin and chymotrypsin.     

The activity of topoisomerase I is modulated by large T antigen during unwinding of the SV40 origin

When simian virus 40 (SV40) large T antigen binds to the virus origin of replication, it forms a double hexamer that functions as a helicase to unwind the DNA bidirectionally. We demonstrate in this report that T antigen can unwind and release an origin DNA single strand of less than full length in the presence of purified human topoisomerase I. The sites nicked by topoisomerase I in the strands released by T antigen during DNA unwinding were localized primarily to the "late" side of the origin, and the template for lagging strand synthesis was preferred significantly over the one for leading strand synthesis. Importantly, these sites were, for the most part, different from the sites nicked by topoisomerase I in the absence of T antigen. These data indicate that T antigen activates topoisomerase I nicking at discrete sites and releases these nicked strands during unwinding. We hypothesize that a single molecule of topoisomerase I can form a functional complex with a double hexamer of T antigen to simultaneously relax and unwind double-stranded origin-containing DNA.     

Transcriptional activation of the human c-myc gene by simian virus 40 large T antigen without binding to p53 and RB proteins in the transient expression system

Male adult Wistar rats were randomly divided into four groups in a 2 x 2 factorial design and were fed diets containing cooked-stored polished rice (CSPR), with and without 0.7 g/100 g of Nebacitin [bacitracinneomycin sulfate (2:1, wt/wt)] and with and without 1 g cholesterol/100 g diet. The CSPR diet contained 1.87 g resistant starch/100 g. After 4 wk, arterial blood and liver were collected. Feces were collected during the last 7 d. Rats fed the diet with Nebacitin and cholesterol had higher serum total cholesterol than the rats fed diets without cholesterol. Serum triglyceride concentration was greater in rats fed Nebacitin, regardless of dietary cholesterol concentration. Rats fed the diet with Nebacitin and cholesterol had higher serum LDL cholesterol concentration and liver total cholesterol concentration than rats fed the other three diets. Rats fed the CSPR diet with Nebacitin both with and without cholesterol had a higher fecal resistant starch concentration and excretion and lower serum short-chain fatty acid concentration than rats fed the diets without Nabacitin. Hepatic cholesterol concentration was greater in rats fed Nebacitin only when the diet also contained cholesterol. Therefore, dietary Nebacitin alters lipid metabolism in rats, and some effects are most pronounced in those also fed cholesterol.     

Amplification of a SV40 T antigen transgene is associated with sarcomagenesis in mice

Transgenic mice harboring simian virus 40 large T antigen (Tag) gene fused to an erythroid-specific enhancer developed soft tissue sarcomas which expressed very high levels of T antigen. The Tag expression was not detectable in the animals' non-transformed tissues. While mice bearing several copies of the transgene developed tumors at an early age of 4-6 months, those with a single copy had a delayed onset of 10-16 months, and DNA analysis of their tumors showed amplification of the Tag transgene. Amplification of a Tag transgene has also been described previously in brain tumors. Our studies demonstrate that Tag transgene amplification is not restricted to a particular construct or a single tumor type. Therefore, this may be a general mechanism for Tag-mediated carcinogenesis, and our transgenic mouse system can be useful for elucidating the mechanisms that govern the amplification process of Tag sequences in vivo.     

The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase alpha-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.     

An epitope on Ki antigen recognized by autoantibodies from lupus patients shows homology with the SV40 large T antigen nuclear localization signal

OBJECTIVE: Epitopes on Ki antigen were analyzed using synthetic peptides, including KILT, a 16-mer peptide with an amino acid sequence homologous to the SV40 large T antigen nuclear localization signal (SV40 T NLS). METHODS: In addition to KILT, 4 synthetic peptides, all potential epitopes on Ki antigen according to computer analysis, were prepared and tested for reactivity with 49 anti-Ki-positive lupus sera by enzyme-linked immunosorbent assay. RESULTS: Eighteen sera reacted with KILT, but not with other peptides. The reaction of anit-Ki sera with KILT was specifically inhibited by recombinant Ki antigen. Eight of 49 anti-Ki sera reacted with a 7-mer synthetic peptide of SV40 T NLS, and the reaction was specifically inhibited by KILT. CONCLUSION: The 16-mer Ki peptide containing the sequence homologous to the SV40 T NLS is one of the antigenic epitopes recognized by anti-Ki antibodies in lupus sera.     

N18-RE-105 cells: differentiation and activation of p53 in response to glutamate and adriamycin is blocked by SV40 large T antigen tsA58

A sodium dodecyl sulphate-agarose apolipoprotein(a) [apo(a)] phenotyping method was set up to attain accurate scanning densitometry of proteins. Serum samples from 99 healthy Spanish men were analysed and twenty-five different apo(a) isoforms (12 to 37 kringle 4 repeats) were detected. Double-band phenotypes accounted for 39.4% (n = 39) and three different patterns of protein expression were identified: pattern A (20.5% of double-band phenotyped samples) predominantly expressed the highest molecular weight isoform; pattern B (53.9%) mainly the lowest molecular weight isoform, and pattern AB (25.6%), expressed both isoforms equally. A significant linear association between expression pattern and lipoprotein(a) [Lp(a)] concentration > or = 0.30 g/l was observed. Single-band phenotyped samples (n = 60) were stratified according to apo(a) kringle 4 repeat categories and showed that 90% of isoforms < 20 K4 repeats had high Lp(a) concentrations (> or = 0.30 g/l), whereas isoforms with 20 to 24 or more than 24 kringle 4 repeats had Lp(a) concentrations > or = 0.30 g/l in 47% and 14%, respectively. A logistic regression model was fitted to test the association between apo(a) size, expression pattern and Lp(a) concentration. In this model, apo(a) isoform < 25 kringle 4 repeats was significantly associated with serum Lp(a) concentration > or = 0.30 g/l in both single and double-band phenotyped samples (odds ratio = 8.9, p < 0.001). In the latter, a differential expression pattern with respect to smaller size isoforms (pattern AB vs A) was significantly associated with Lp(a) concentration > or = 0.30 g/l (odds ratio = 17.97, P = 0.045). Heterogeneity in protein apo(a) size expressed according to kringle 4 repeat number could be categorized in heterozygous phenotypes as three patterns. When small-sized isoform was expressed (pattern B) or both isoforms were equally expressed (pattern AB), the probability of having Lp(a) > or = 0.30 g/l is higher.     

The J domain of simian virus 40 large T antigen is required to functionally inactivate RB family proteins

Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We have previously demonstrated that the N-terminal J domain of TAg affects the RB-related proteins by perturbing the phosphorylation status of p107 and p130 and promoting the degradation of p130 and that this domain is required for transformation of cells that express either p107 or p130. In this work, we demonstrate that the J domain of TAg is required to inactivate the ability of each member of the pRB family to induce a G1 arrest in Saos-2 cells. Furthermore, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. These results imply that while the LXCXE domain serves as a binding site for the RB-related proteins, the J domain plays an important role in inactivating their function.     

T cells are responsive to the simian virus 40 large tumor antigen transgenically expressed in pancreatic islets

The qualities of a peripheral Ag that determine whether T cells will be tolerant of or responsive to it are poorly understood. To approach this problem, we studied the T cell response in a line of transgenic mice selectively expressing an oncoprotein in the islets of Langerhans. The SV40 large tumor Ag (SV40-T) is directed to islet beta-cells in Rip1-Tag3 (RT3) mice by a hybrid insulin promoter-SV40-T construct. Ag is first detected on these cells between 10 and 12 wk after birth. RT3 mice were bred with mice expressing a transgenic rearranged TCR recognizing SV40-T in the context of the class I MHC molecule, H-2Kk. T cell response in the resultant RT3/TCR-double transgenic mice was then analyzed. T cells are fully responsive to SV40-T in RT3/TCR-transgenic mice, and T cells infiltrate the islets of both RT3 and RT3/TCR-transgenic mice. This work demonstrates that T cells may remain responsive to self-Ag expressed outside the thymus, and that this responsiveness may result in autoimmunity. The developmentally delayed expression or the oncogenic nature of SV40-T in the RT3-transgenic mice may be important in determining this T cell response.     

Genomic imprinting and Igf2 influence liver tumorigenesis and loss of heterozygosity in SV40 T antigen transgenic mice

Maternal-specific loss of heterozygosity (LOH) and allelic imbalances [i.e., partial LOH (pLOH)] observed in SV40 T/t antigen-induced liver tumors suggests that an imprinted gene on chromosome 7 is involved in liver tumorigenesis. Maternal-specific LOH/pLOH may reflect the loss of a maternally expressed tumor suppressor gene or the acquisition of paternally active alleles of a growth promoter. In addition, two oppositely imprinted genes on distal chromosome 7, Igf2 and H19, are re-expressed in most liver tumors from an SV40 T/t antigen transgenic line (M11T-G). Igf2 is a paternally expressed growth promoter, and H19 is a maternally expressed gene that can suppress growth in some tumor cell lines. We studied the role of Igf2 during liver tumorigenesis by creating Igf2 (+/-) M11T-G mice. These mice are essentially null for Igf2 expression because imprinting normally precludes maternal Igf2 expression. M11T-G, Igf2 (+/-) males exhibit a 15-fold reduction in the frequency of large tumors. Igf2 (+/-) tumors do not express maternal Igf2, indicating rigid imprinting control in the liver. LOH/pLOH analysis was performed on the tumors and indicates that acquisition of paternally active Igf2 alleles is a major selective event for M11T-G liver tumorigenesis. This also implies the existence of an imprinted, maternally expressed tumor suppressor gene on chromosome 7 that is unlikely to be H19.