Interleukin 12 mediated prevention of tumorigenicity in murine cell lines derived from CD40L transgenic mice

Cells derived from superficial and deep lymph nodes of transgenic mice in which CD40L expression was deregulated were grown in vitro. After 3 months of interleukin 3 or interleukin 12 stimulation, the cells remained interleukin-independent, showed the same in vitro growth characteristics, but LIL3+ cells were tumorigenic when reinoculated in vivo in nude mice, whereas interleukin-12-treated cells did not induce tumors. Our cell lines could provide a useful model to study the perturbation of the homeostasis allowing us to elucidate the role of cytokines as modulators of differentiation in the lymphoproliferative disorders.     

Frequency of SV40-specific cytotoxic T-lymphocyte precursors in two SV40 T-antigen transgenic mouse lines.

Cytotoxic T-lymphocyte precursors (CTL-P) from control C57BL/6 mice and from mice of two simian virus 40 (SV40) T-antigen transgenic lines, 427 and 419, specifically nonresponsive and responsive, respectively, to SV40 T antigen, were quantitated by limiting dilution (LD) after immunization with SV40. CTL-P frequencies for the SV40 T antigen-responsive 419 line transgenic mice were within the range established in C57BL/6 mice, whereas no CTL-P could be demonstrated for the SV40 T antigen-tolerant 427 line mice. These results suggest that deletion or anergy of SV40 T antigen-responsive clones underlies the specific profound tolerance of 427 line mice.     

Establishment of transformed swine fibroblast cell lines using SV40 large T antigen

Summary Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.     

Cytogenetic findings in cell lines derived from four ovarian carcinomas

Six cell lines, established from four primary ovarian carcinomas were examined cytogenetically. The lines varied greatly in their chromosome complement. All cells from the lines were aneuploid, although one cell line contained two populations having a pseudodiploid and a pseudotetraploid modal chromosome number. Every chromosome group was involved with loss and gain of chromosomes, but some individual chromosomes were more prone to aneuploidy than others. Chromosome #6 was the most stable throughout. Structural changes gave rise to many marker chromosomes. Although most markers were random and the majority unidentifiable, some abnormalities of clonal origin were found. Deletions especially of chromosome #1, were the most common change. Further sequential studies may elicit the origin, stability, and timing of the chromosome abnormalities.     

Cytogenetic analysis of SV40-transformed human breast epithelial cells

The SV40-transformed breast epithelial cell lines established by Chang et al. [1] were shown to be hypotetraploid and characterized by six chromosome markers: M1 i(1q), M2 del(1)(q21), M3 i(6p), M4 del(1)(q11), M5 t(8p;12q), and M6 dir dup(11)(p12→pter). The presence of common chromosome markers indicates that these cell lines are probably derived from the same original transformed cell.     

Specific DNA binding activity of T antigen subclasses varies among different SV40-transformed cell lines

Large tumor antigen (T antigen) occurs in at least three different oligomeric subclasses in cells infected or transformed by simian virus 40 (SV40): 5-7 S, 14-16 S, and 23-25 S. The 23-25 S form is complexed with a host phosphoprotein (p53). The DNA binding properties of these three subclasses of T antigen from nine different cell lines and free p53 protein were compared using an immunoprecipitation assay. All three subclasses of T antigen bound specifically to SV40 DNA sequences near the origin of replication. However, the DNA binding activity varied between different cell lines over a 40- to 50-fold range. The 23-25 S and 14-16 S forms from most of the cell lines tested bound much less SV40 origin DNA than 5-7 S T antigen. The free p53 phosphoprotein did not bind specifically to any SV40 DNA sequences.     

Analysis of the nonviral antigens immunoprecipitable by SV40 T antibody from SV40-transformed human/mouse hybrid cell lines

A temperature-sensitive (ts) mutant of herpes simplex virus type 1 (HSV-1), tsJ12, is able to undergo one cycle of replication at the nonpermissive temperature (39°) yielding wild-type quantities of enveloped virus particles. These particles contain viral DNA which is as infectious as wild-type viral DNA; however, they are not infectious. Analysis of [¹⁴C]glucosamine-labeled mutant-infected cell extracts by one- and two-dimensional polyacrylamide gel electrophoresis demonstrated that at 39° tsJ12 fails to induce the synthesis of both the mature gB glycoprotein and its dimeric form which are normal constituents of the virion envelope. Polyethylene glycol, an agent which promotes membrane fusion, enhances the infectivity of tsJ12 virions by greater than 1000-fold following adsorption of virus to susceptible cells demonstrating that mutant virions are able to attach to cells but not penetrate. Consistent with a defect in the virion envelope, tsJ12 is able to interfere with the production of infectious wild-type virus, presumably by the formation of pseudotypic virions composed of wild-type viral genomes in gB-deficient envelopes. Physical mapping of the is defect in this mutant demonstrates that it lies within the limits of the DNA sequence which specifies gB on the physical map of the genome. A ts⁺ revertant of tsJ12 is as infectious as wild-type virus and synthesizes a gB glycoprotein which is indistinguishable from that of wild-type virus. Thus, biological and biochemical studies of tsJ12 and of a ts+ revertant of this mutant (1) demonstrate that glycoprotein gB is essential for infectivity at the level of penetration and (2) further define the physical map location of the gene for this glycoprotein.     

Tumorigenic and metastatic ability of SV40-transformed BALB/c cell lines and MHC antigen expression

Tumorigenic and metastatic potential were studied in relation to class I MHC expression in four different SV40-transformed BALB/c cell lines. All the lines studied, tumorigenic or not, expressed both H-2Kd and Dd, so MHC antigens did not seem to be involved in the control of SV40-transformed cells' growth in vivo. Lung metastases were observed in all tumour-bearing mice. Cells cultured after in vivo passage, obtained either from tumour tissue or from individual lung metastases, still expressed similar levels of H-2d antigens, thus suggesting that tumour growth and metastasis do not occur through the selection of variants with altered MHC expression.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). VI. Mechanism of induction of SV40 transplantation immunity in mice by purified SV40 T antigen (D2 protein)

According to the base sequence homology of the gene coding for the nonstructural (NS) protein the influenza A viruses can be divided into at least three groups. Within each group the homology is about 90% or higher. The avian influenza A viruses fall at least into two groups between which the homology is about 40%. All human strains tested belong to another group. Influenza viruses of other species might comprise their own group(s). The related regions of the NS gene among the two groups of avian influenza viruses overlap completely and they are highly conserved. The results are discussed in terms of a selection pressure concerning the function exerted by the host during the evolution of the different NS genes, which also might explain a certain species specificity concerning the NS gene of influenza A viruses.     

Phosphorylation of SV40 large T antigen in SV40 nucleoprotein complexes

Plaques produced by our P^? mutants of vesicular stomatitis virus (VSV), which are defective in the inhibition of total protein synthesis in infected cells, stop increasing in size after several days of incubation under conditions where those produced by P⁺ mutants increase linearly in size. The basis for the arrest in size of P^? plaques has been shown to be due to the induction of interferon, and the phenotype is termed PIF⁺ for “plaque interferon positive.” Thus P^? plaques can inhibit the increase in size of adjacent P⁺ plaques and the factor responsible has the biological and physical properties of interferon. Also P^? mutants, when plaqued on VERO cells which cannot be induced to produce interferon, produce plaques which increase linearly in size like P⁺ plaques. Finally, the inclusion of anti-interferon antibody in the overlay medium also causes P^? mutants to produce plaques like P⁺ mutants. VERO cells were found to be useful to separate the effects of is mutations on plaque size from the interferon effect. Using other cell types the latter effect (PIF assay) can be used as an assay for the ability of viruses to induce interferon, for the isolation of PIF⁺ mutants from PIF^? virus, and as a test for the ability of cells to respond to interferon induction by PIF⁺ viruses. The assay can be increased in sensitivity through the use of specific cell types and of cell cultures preincubated for several days in the stationary phase of growth. In its most sensitive form, the assay could detect PIF⁺ behavior in certain ts mutants of VSV at permissive temperatures and in VSV mutants emerging from persistent infection. The assay has also been used to isolate novel mutants of VSV which show alterations in the viral P function.     

Studies on the SV40-like papovavirus SV40-GBM. III. Propagation at low multiplicities of infection in various human cell lines

At low multiplicity several human cell lines supported the lytic infection with SV40-GBM better than that with the wild type SV40. The efficiency of viral DNA replication differed in the cell lines used suggesting that specific host cell factors may determine the rate of viral DNA synthesis. Furthermore, the emergence of different DNA defects during propagation of the virus indicates that host cell factors in question might also influence the composition of the viral DNA population.     

Characterization of defective SV40 isolated from SV40-transformed cells.

Defective SV40 viruses were isolated from SV40-transformed monkey, human and hamster cells after Sendai virus-mediated fusion of the transformed cells with TC7 cells, a stable line of African green monkey kidney cells. Viral isolates were concentrated and purified and the defective viruses examined by electron microscopy. The buoyant densities in CsCl of the defective viruses ranged between 1.32 and 1.33 g/cc. DNA isolated from defective viruses was characterized by dye-buoyant density centrifugation and by velocity sedimentation in neutral CsCl. The DNA was heterogeneous in size and contained some covalently closed double-stranded circular molecules.     

Contact-inhibited revertant cell lines isolated from SV40-transformed cells. VIII. Membrane protein and glycoprotein composition.

The composition of enriched surface membrane preparations from BALB/c 3T3, SV40-transformed 3T3 (SVT2), and concanavalin A-selected revertant variant cells were investigated by polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate. The electrophoretic profiles of the membrane proteins from the three cell lines were remarkably similar. However, [¹⁴C]leucine-radiolabeled or Coomassie blue-stained preparations from revertant cells lacked a protein band of apparent molecular weight 110,000 (band 15), which was present in 3T3 and SVT2 membranes and which was replaced in revertant membranes by two new bands (15a and 15b) migrating on either side of band 15. Lactoperoxidase-catalyzed iodination studies indicated that the relative proportion of band 5 (a LETS-like glycoprotein) was decreased, and band 24 (MW, 65,000) was increased in 3T3 cells compared to the other cell lines. Revertant membranes had increased proportions of sialic acid residues on several glycoproteins. The LETS glycoprotein from all three cell types was found to be sialylated. Two different compartmentalized pools of cell surface-associated LETS have been identified. These results are discussed in relation to the method of selection of revertant cells and to the possible changes induced in cell surface architecture by transformation and reversion.     

Tumorigenesis in transgenic mice by a nuclear transport-defective SV40 large T-antigen gene

The SV40(cT) mutant encodes a large tumor antigen (cT-ag) that is defective for transport from the cell cytoplasm into the nucleus. This mutant is able to transform established cell lines at near wild-type virus efficiencies, but has a markedly decreased ability to transform primary cells and to induce tumors in newborn hamsters (R. E. Lanford, C. Wong, and J. S. Butel, 1985, Mol. Cell. Biol. 5, 1043-1050). To explore the biology of transport-defective T-ag in vivo, transgenic mice carrying the cT-ag gene were produced. Five of eight founder animals died early in life of choroid plexus tumors (mean age +/- SE, 52 +/- 11.0 days); renal and thymic lesions were also observed. Mice of an SV40(cT) transgenic line regularly succumb to brain tumors (mean age, 81 +/- 1.2 days). SV40 T-ag is expressed in the tumor cells and is retained in the cytoplasm. The observation that SV40(cT) is equivalent to wild-type virus at tumor induction in transgenic mice emphasizes the probable importance of extranuclear forms of SV40 T-ag in brain tumor formation. This study also indicates that in vitro cell transformation assays may not always be accurate reflections of the oncogenic potential of a transforming gene in vivo, because of the different cell types involved.     

Critical role for SV40 small-t antigen in human cell transformation

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.     

Cell lines produced from tumors caused by virus SV 40

Summary A transplantable cellular strain, CA-SV 40-63-1, was produced from a sarcoma in a Syrian hamster induced by injection of SV 40 virus. The cellular strain formed after a prolonged lag-phase. The CA-SV 40-63-1 cultures were characterized by a marked cellular polymorphism, high mitotic activity, a tremendous number of giant mono- and polynuclear cells. The administration of cells of the CA-SV 40-63-1 strain to Syrian hamsters causes the formation of sarcomas of which the cellular composition bears a striking resemblance to that of the CA-SV 40-63-1 strain. One of such tumors gave rise to a new cellular strain, CA-SV 40-63-1 bis, which formed without the lag-phase. The cytological characteristics of the 2nd cellular strain are close to those of the CA-SV 40-63-1 strain. Certain aspects of the problem of relationships between the processes of malignization of cells in vivo and transformation of cells in vitro are also discussed.Presented by Active Member of AMN SSSR V. M. Zhdanov Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 59, No. 5, pp. 85–88, May, 1965