Assignment of the T-antigen gene of simian virus 40 to human chromosome C-7

Hybrid cell clones between mouse cells deficient in thymidine kinase (EC 2.7.1.21) and two different human cell lines transformed by simian virus 40 (SV40) and deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) were examined for SV40 tumor (T) antigen(s). Concordant segregation of the gene(s) for SV40 T antigen and human chromosome C-7 was observed in these hybrids. The human chromosome C-7 which contains the gene(s) for SV40 T antigen is preferentially retained by the majority of the hybrid clones tested. When hybrid clones positive and negative for SV40 T antigen, derived from the fusion of SV40-transformed Lesch-Nyhan fibroblasts with mouse cells, were fused with CV-1 permissive cells, SV40-specific V antigen was observed only in the cultures derived from fusion of the hybrid clones positive for T antigen. This result indicates a linkage relationship between human chromosome C-7, SV40 T-antigen gene(s), and SV40 genome(s) integrated in the human transformed cells.     

Another chromosomal assignment for a simian virus 40 integration site in human cells.

Somatic cell hybrids derived from fusion of GM637, a human cell line transformed by simian virus 40, and mouse B82 cells were examined for simian virus 40 T antigen, V antigen, and viral DNA. All hybrid cell lines that contained viral DNA were T-antigen positive. Cells that did not have viral DNA were T-antigen negative. We determined that there is a single viral insertion in these hybrid cells. Correlation of T-antigen expression and viral DNA with the partial complements of the human genome retained in the hybrids shwed that the inserted viral genome is in human chromosome 8. The integrated viral DNA is stable; free viral DNA found in GM637 does not insert at other potential sites in the human genome.     

Transformation-related antigens identified by monoclonal antibodies.

Tumor-cell proteins that were antigenic in a syngeneic animal were identified by immunoprecipitation with monoclonal antibodies. Spleen cells of BALB/c mice immunized with plasma membranes of Kirsten RNA sarcoma virus-transformed BALB/3T3 cells were fused with NS-1 myeloma cells. Antibodies secreted into the culture fluid from these hybridomas were distinguished by their reactivity against proteins of different target cells. A total of 191 cultures were established; 143 produced antibodies that bound to BALB/3T3 cells transformed by the RNA sarcoma virus, of which antibodies from 82 bound to BALB/3T3 transformed with simian virus 40, and antibodies from 56 bound to BALB/3T3 cells. Thus, more than 50% of the cultures produced antibodies that possibly were specific to antigens of the transformed cell. Twenty different hybridomas have been cloned, and antibodies, from eight of these were found to immunoprecipitate five different proteins. A protein of approximately 32,000 daltons was precipitated from BALB/3T3 cells transformed by the RNA sarcoma virus, simian virus 40, or methylcholanthrene but not from untransformed BALB/3T3 cells. A protein of about 300,000 daltons was precipitated from all four cell lines; precipitation was enhanced in the viral transformed cells. Proteins of approximately 57,000, 54,000, and 8500 daltons were immunoprecipitated from all four cell lines.     

Adenovirus 2 early gene expression promotes susceptibility to effector cell lysis of hybrids formed between hamster cells transformed by adenovirus 2 and simian virus 40.

Weakly oncogenic adenovirus 2 (Ad2)-transformed LSH hamster cells are sensitive to lysis by spontaneously cytolytic lymphoid cells and activated macrophages, whereas highly oncogenic simian virus 40 (SV40)-transformed LSH cells are relatively resistant to these nonspecific effector cells. Somatic cell hybrids formed between Ad2- and SV40-transformed hamster cells, which expressed Ad2 tumor (T) antigens, exhibited an increased cytolytic susceptibility compared to Ad2 T antigen-negative cell hybrids or nonhybrid SV40-transformed cells. No correlation was found between the expression of SV40 T antigen in hybrid cells and cytolytic susceptibility. The results suggest the existence of a novel function for early Ad2 genome-encoded polypeptides (T antigens) expressed in transformed hamster cells--the induction of susceptibility to destruction mediated by immunologically nonspecific effector cells.     

Production of simian virus 40 large tumor antigen in bacteria: altered DNA-binding specificity and dna-replication activity of underphosphorylated large tumor antigen.

A bacterial expression system was used to produce simian virus 40 large tumor antigen (T antigen) in the absence of the extensive posttranslational modifications that occur in mammalian cells. Wild-type T antigen produced in bacteria retained a specific subset of the biochemical activities displayed by its mammalian counterpart. Escherichia coli T antigen functioned as a helicase and bound to DNA fragments containing either site I or the wild-type origin of replication in a manner identical to mammalian T antigen. However, T antigen purified from E. coli did not efficiently bind to site II, an essential cis element within the simian virus 40 origin of replication. It therefore could not unwind origin-containing plasmids or efficiently replicate simian virus 40 DNA in vitro. The ability of protein phosphorylation to modulate the intrinsic preference of full-length T antigen for either site I or site II is discussed.     

Homologous recombination between a defective virus and a chromosomal sequence in mammalian cells.

Replacement of the early region of simian virus 40 results in virus that cannot replicate in a normal host, CV-1 cells, but can replicate in COS cells, a derivative of CV-1 cells that constitutively express simian virus 40 tumor antigen (T antigen). However, passage of such an early replacement simian virus 40 mutant in COS cells results in the emergence of virus that can propagate in CV-1 cells. Analysis of this virus revealed that the mutant rescued the integrated T-antigen gene from the COS cell genome. Comparison of the sequence of the recovered virus with that of the viral DNA resident in COS cells (strain 776) and the mutant used in our studies (derived from strain 777) proves that the mutant virus acquired the T-antigen gene from the COS cell chromosome via homologous recombination. Most probably this process was mediated by a direct genetic exchange.     

Transformation by Simian Virus 40 of Spleen Cells from a Hyperimmune Rabbit: Evidence for Synthesis of Immunoglobulin by the Transformed Cells

Spleen cells derived from a rabbit hyperimmunized with a Type III pneumococcal vaccine were exposed to simian virus 40 in vitro. After 114 days, transformed cells growing on the surface of one culture dish were observed and a cell line was established. The transformed cells had a morphology characteristic of cells transformed by simian virus 40, contained the simian virus 40-specific T antigen, and yielded infectious simian virus 40 upon cultivation with indicator cells in the presence of Sendai fusion factor. Transformed cells incorporated labeled amino acid into protein with the antigenic properties of rabbit immunoglobulin G.     

Simian virus 40-transformed adherent cells from human long-term marrow cultures: cloned cell lines produce cells with stromal and hematopoietic characteristics

Adherent cells from long-term marrow cultures from 23 individuals were transformed with wild-type simian virus 40 (SV40). After transformation, cloned cell lines were developed that even after rigorous subcloning invariably produced both stromal cells and round cells. The stromal cells expressed cytoskeletal filaments similar to those of long-term marrow culture adherent cells and produced interstitial and basal lamina collagen types. The round cells had the electron microscopic appearance of primitive hematopoietic cells and when examined with cytochemical stains and monoclonal antibodies to hematopoietic differentiation antigens had reaction patterns suggestive of cells from several lineages. Most round cells expressed the pan- hematopoietic T-200 determinant, and lesser percentages expressed the early T cell antigens CD-1 and CD-3, HLA-DR determinants, the monocytic antigen recognized by Leu M3, and the myeloid antigens detected by monoclonal antibodies 1G10 and 12.8. In addition, when plated in semisolid medium in the presence of a source of colony-stimulating activity, up to 11% of the cells formed colonies consisting of blastlike cells that also expressed hematopoietic cell surface determinants. The data suggest that adherent cells in long-term marrow cultures contain a cell that after transformation by SV40 obligately produces cells with hematopoietic as well as stromalike features.     

Extinction of JC virus tumor-antigen expression in glial cell--fibroblast hybrids.

JC virus (JCV) is a ubiquitous human papovavirus that shares sequence and structural homology with simian virus 40 (SV40). In contrast to SV40, expression of JCV is restricted to a small number of cell types, including human fetal glial cells, uroepithelial cells, amnion cells, and some endothelial cells. To study the control of JCV early region expression, we made heterokaryons and stable hybrids between JCV-transformed hamster glial cells and mouse fibroblasts. Binucleate heterokaryons exhibited extinction of large tumor antigen expression in the hamster nuclei as assayed by indirect immunofluorescence. This extinction was both time and dose dependent: extinction reached maximal levels at 24-36 hr after fusion and was dependent on the ratio of glial cell to fibroblast nuclei in multinucleated heterokaryons. Extinction also was observed in stable hybrids between the glial cells and mouse Ltk- cells. Southern blot analysis showed that the extinguished hybrids contained viral sequences. Reexpression of large tumor antigen was observed in several subclones, suggesting that extinction was correlated with the loss of murine fibroblast chromosomes from these hybrids. The cis-acting region that mediates extinction resides within the viral regulatory region, which contains two 98-base-pair repeats that have enhancer activity. These data demonstrate that cellular factors that negatively regulate viral gene expression contribute to the restricted cell-type specificity of this virus.     

Overproduction of the protein product of a nonselected foreign gene carried by an adenovirus vector.

We have constructed a recombinant adenovirus that carries the herpes simplex virus type I gene for thymidine kinase (EC 2.7.1.21) and expresses thymidine kinase under control of adenovirus major late promoter. A DNA fragment carrying thymidine kinase coding sequences but lacking the thymidine kinase promoter was sandwiched between a piece of adenoviral DNA and simian virus 40 early DNA on a plasmid. The aligned fragment was then inserted into the adenoviral genome, replacing internal adenoviral DNA. Hybrid viruses carrying the thymidine kinase gene were obtained by selecting for viruses that express simian virus 40 tumor antigen (T antigen) in monkey cells. The thymidine kinase gene was positioned in the third segment of the adenovirus tripartite leader downstream from the major late promoter by in vivo DNA recombination between the duplicated adenoviral sequences present in the plasmid insert and the viral vector. Levels of thymidine kinase activity in human or monkey cells infected with this hybrid virus were several times higher than in cells infected with herpes simplex virus. Infected cells produced thymidine kinase protein at very high levels, similar to those found for adenovirus late major capsid proteins. The thymidine kinase protein represented 10% of the newly synthesized protein in late infected cells and accumulated to represent 1% of total cell protein under optimal conditions. This vector system offers a procedure by which a variety of gene products that are biologically active and properly modified can be produced at high levels in mammalian cells.     

Biological activity of purified simian virus 40 T antigen proteins.

Proteins related to simian virus 40 (SV40) T antigen uere isolated from cells infected with adenovirus 2/SV40 hybrids Ad2+D2 and Ad2+ND1 dp2 as well as from a line of human cells (SV80) transformed by SV40. The 96,000- and 107,000-dalton proteins of SV80 and Ad2+D2, after injection into the cytoplasm of cultured cells, rapidly accumulate in the nuclei, where they remain antigenically reactive for at least 20 hr and trigger DNA synthesis in quiescent cells. By contrast, the 23,000-dalton protein coded by Ad2+ND1 dp2 does not stimulate cellular DNA synthesis. However, all three purified proteins are able to provide helper function for the growth of adenovirus 2 in monkey cells. Thus, purified SV40 T antigen and proteins that share sequences with it retain the ability to carry out at least two functions associated with the product of the A gene of SV40.     

Assignment of gene(s) for cell transformation to human chromosome 7 carrying the simian virus 40 genome.

Somatic cell hybrid clones between either C57BL/6 or Balb/c mouse peritoneal macrophages and two different simian virus 40 (SV40)-transformed human cell lines deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferase) were obtained in hypoxanthine-aminopterin-thymidine selective medium. All the hybrid cell clones contained the human chromosome 7, which carries the SV40 genome, and were SV40 tumor (T)-antigen positive. No hybrid cell clones studied displayed the density-dependent inhibition of cell growth characteristic of normal cells; all clones had a high saturation density and gave origin to cell colonies when plated in soft agar. Since the expression of the transformed phenotype was always associated with the presence of the human chromosome 7, which carries the SV40 genome, it is concluded that this chromosome contains gene(s) [Tr gene(s)] coding for "transforming factor(s)."     

Expression of human class II major histocompatibility complex antigens using retrovirus vectors

Retrovirus vectors [direct orientation (DO) vectors] that permit the simultaneous expression of an inserted protein-coding sequence and a dominant-acting selectable marker have been constructed. In these vectors, an internal simian virus 40 or human metallothionein promoter sequence serves to drive the expression of the bacterial neomycin phosphotransferase or guanine-xanthine phosphoribosyltransferase genes, whereas the viral long terminal repeat sequences are utilized to promote expression of inserted sequences. In some of the vectors, the viral 5' splice site, normally used in the biogenesis of the subgenomic env-encoding mRNA, has been eliminated. These vectors yield high transient and stable titers of virus after transfection of viral packaging cell lines, show little or no depression of virus titer with a variety of inserts, and faithfully transmit recombinant proviral sequences to recipient cells. To characterize the expression potential of these vectors, a variety of inserts encoding the alpha and beta subunits of the human major histocompatibility complex class II antigen HLA-DR have been introduced into these vectors. NIH 3T3 cells infected by viruses containing HLA-DR alpha or beta cDNAs express these proteins as shown by immunoprecipitation of metabolically labeled extracts. In addition, through the sequential infection of cells with retrovirus constructions expressing two different selectable markers, both subunits of the class II antigen have been introduced into NIH 3T3 cells. Such infected cells express HLA-DR molecules at the cell surface.     

Resistance of human cells to tumorigenesis induced by cloned transforming genes.

The transformation of human cells was examined by transfection of cloned oncogenic DNAs derived from the tumor virus simian virus 40 and from the human bladder carcinoma cell line EJ into diploid fibroblasts derived from foreskin (FS-2 cells). The simian virus 40 DNA was found to induce a morphologically transformed phenotype, leading to easily detectable focus formation. Tumor antigen was produced, but the transformed cells were not tumorigenic in the nude mouse. The EJ gene, a mutant form of the cellular c-Ha-ras gene, actively transforms NIH/3T3 mouse cells and CHEF/18 hamster cells but is inactive in FS-2 cells. Morphological transformation, focus formation, and tumorigenicity in nude mice were not induced when EJ DNA was transfected into FS-2 cells by using the selectable vector pSVgptEJ. The intactness of the transfected EJ DNA was established by restriction fragment analysis. This result raises the question of what role, if any, the mutated gene derived from the EJ cells played in the origin of the EJ bladder carcinoma.     

Suppression of tumor growth by senescence in virally transformed human fibroblasts.

Normal human cells whether embryonic, neonatal, or adult are resistant to experimentally induced tumorigenesis in contrast to rodent or chicken cells. We showed previously that neither transformation with simian virus 40 DNA nor transfection with human mutant HRAS DNA immortalized FS-2 cells (diploid, neonatal human fibroblasts). Further, tumorigenicity was not induced, despite expression of the respective transforming gene products tumor (T) antigen or p21. Here we describe treatment of FS-2 and FSSV cells with baboon endogenous virus pseudotyped Kirsten murine sarcoma virus. FSSV cells were derived from individual foci of simian virus 40-transformed FS-2 cells. The retrovirus-treated FS-2 cells (called FSK) appeared heavily granulated and expressed viral p21 but senesced during passage in culture and were not tumorigenic. The retrovirus-treated FSSV-27 cells (called FSVK-27) expressed simian virus 40 tumor antigen, had elevated levels of viral p21 protein, and formed transient tumors in nude mice. Whether grown in culture or explanted from small tumors, the FSVK-27 cells senesced. The FSVK-46 cells senesced before tumor growth occurred. On the contrary, Kirsten murine sarcoma virus (baboon endogenous virus) treatment of immortalized nontumorigenic human fibroblasts expressing simian virus 40 tumor antigen (Va2 cells) led to consistent tumor formation. The results illustrate the importance of senescence in restricting the tumor-forming ability of human cells.     

Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene.

Established REF52 cells (rat embryo fibroblasts) completely resist stable transformation by ras oncogenes, and simian virus 40 large tumor (T) antigen collaborates with ras to convert REF52 cells to tumorigenic state. A temperature-sensitive simian virus 40 large T antigen (encoded by tsA58) allowed the T24 Ha-ras oncogene to transform REF52 cells in a temperature-dependent manner. Two thirds of the clones transformed with tsA58 and ras became arrested in G2 or late S phase when shifted to a nonpermissive temperature for T antigen stability. Thus, ras induced growth arrest rather than stable transformation in the absence of a functional collaborating oncogene. These results indicate that collaborating oncogenes can regulate cellular responses to ras and have implications regarding therapeutic strategies to control tumor cells expressing activated ras oncogenes.     

Immunologic selection against simian virus-40 transformed cells: Concomitant loss of viral antigens and early viral gene sequences

A clonal line of highly oncogenic "spontaneously transformed" mouse cells (T AL/N clone 3) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned. The clone of SV40-transformed cells (subclone 1) expressed SV40-specific T (nuclear) and transplantation antigens but was 100 times less tumorigenic than the parent T AL/N clone 3 cells. When large numbers of subclone 1 cells (10(4)-10(5)) were injected into syngeneic AL/N mice, tumors were produced. From the tumors, cell lines were established in culture, all of which were consistently negative for T antigen. Tumor lines tested were found not to contain SV40-specific transplantation antigen and had again become highly tumorigenic. The original subclone 1 cells contained about one copy of SV40 DNA per diploid amount of cell DNA, as well as RNA complementary to the early region of the SV40 genome. The T antigen-negative cells from tumor line 124 contained approximately 0.5 copy of SV40 DNA per diploid equivalent and did not synthesize any detectable virus-specific RNA. Reassociation kinetic analysis with restriction enzyme fragments of viral DNA demonstrated that the cells from tumor line 124 (and also the clones of this line) had lost DNA sequences predominantly from the early region of the SV40 genome. The results indicate that a set of stably integrated SV40 DNA sequences can be present in a cell without the expression of viral antigens.     

Expression of simian virus 40 large T (tumor) oncogene in mouse chondrocytes induces cell proliferation without loss of the differentiated phenotype.

We have infected primary embryonic mouse limb chondrocytes with a retrovirus carrying simian virus 40 early regions and have obtained a monoclonal mouse chondrocyte line, MC615, that was able to grow on culture dishes for at least 7 months and 20 passages. MC615 cells show expression of simian virus 40 large T (tumor) antigen and express markers characteristic of cartilage in vivo, such as types II, IX, and XI collagen, as well as cartilage aggrecan and link protein. These data show that cell growth induced by large T oncogene expression does not prevent the maintenance of the chondrocytic phenotype.     

Studies of the locus for androgen receptor: localization on the human X chromosome and evidence for homology with the Tfm locus in the mouse.

We have established a cell line from mouse kidney cells expressing the tfm mutation and showed that these cells lack androgen binding activity. A subclone of these simian virus 40 (SV40)-transformed cells (6TGR-SV-tfm) selected in 6-thioguanine and lacking hypoxanthine phosphoribosyltransferase was used to produce a series of mouse--human hybrids containing the normal human X chromosome or various X autosome-translocation chromosomes (expressing only segments of the human X chromosome). When the androgen receptor locus (AR) was present in the hybrid, the number of receptor sites and kinetics of binding were similar to that in the human parental cells. Analysis of hybrids with partial human X chromosomes by using assays for X chromosome-linked enzymes and for the androgen receptor protein indicate that the AR locus on the human X chromosome is near the centromere between Xq13 and Xp11 and is proximal to the locus for phosphoglycerate kinase. Hybrids derived from 6TGR-SV-tfm mouse cells and human labial fibroblasts from an XY individual with the ar- form of androgen insensitivity have no binding activity. The lack of complementation indicates that the X chromosome-linked mutations in mouse and man affect homologous loci and supports the evolutionary conservation of X chromosomal loci in mammals; however, the position of the locus on the human X chromosome indicates that intrachromosomal rearrangement has occurred.     

Deletion of 43 amino acids in the NH2-terminal half of the large tumor antigen of simian virus 40 results in a non-karyophilic protein capable of transforming established cells.

We have characterized a simian virus 40 (SV40) mutant, derived from the viral DNA insertion present in simian cell transformants, which carries a deletion affecting the NH2-terminal region of the SV40 large tumor antigen. This mutant protein is 6% smaller than normal, has lost the typical nuclear localization of the SV40 large tumor antigen, and accumulates in the cytoplasm. The deletion begins at nucleotide position 4490 of the SV40 DNA and ends in-frame at nucleotide position 4362. The missing 43 amino acids begin with proline-110 and end with serine-152 of the predicted sequence; they include a cluster of basic residues, presumably important for the viral origin-DNA binding, and most of the phosphorylation sites present in the NH2-terminal half of the molecule. The protein can still be phosphorylated considerably in vivo. This mutant viral genome is replication-defective but has conserved the competence to transform established cells, such as NIH/3T3 cells. Transfection of cloned mutant DNA into such cells resulted in the production of full transformants. Full transformants were not produced in similar transfections carried out in primary rat embryo fibroblasts, although some primary transfectants expressing the non-karyophilic large tumor antigen might be considered minimally transformed.