Lymphotropic papovavirus transforms hamster cells without altering the amount or stability of p53.

Expression of the early regions of several primate polyomaviruses (SV40, BKV, JCV, and LPV) in hamster cells induces transformation, manifested by the ability to grow in soft agar. Hamster cells transformed by SV40 contain complexes between the SV40 T antigen and the cellular tumor suppressor protein p53. We detected analogous complexes between p53 and the BKV T antigen in hamster cells transformed by the BKV early region, where the half life of p53 increased 16-fold. However, neither a LPV-transformed hamster fibroblast cell line [LPV-HE (F); K. K. Takemoto and T. Kanda, 1984, J. Virol. 50, 100-105] nor BHK-21 cells transformed by the LPV early region contained detectable complexes between the LPV T antigen and p53, nor was the stability of p53 in LPV transformed BHK-21 cells altered. Association between hamster p53 and the LPV T antigen expressed as glutathione S-transferase fusion protein could not be detected in vitro. These data indicate that alteration of the amount or stability of p53 is not required for transformation of hamster cells by LPV. However, as viruses such as SV40 and BKV whose T antigens bind p53 are oncogenic in hamsters, whereas LPV is not, the alteration of p53 amount or stability may be required for tumorigenesis.     

Transformation of mouse mammary epithelial cells by papovavirus SV40

Primary and established murine mammary epithelial cells and wild-type SV40 were employed to study the phenomenon of epithelial cell transformation. Thirteen independent transformed cell lines were derived. All contained SV40 intranuclear T antigen. Eight transformed mammary cell lines were examined ultrastructurally and all were found to exhibit pronounced epithelial cell characteristics, including desmosomes and tight junctions. Growth studies revealed that while normal mammary cells were unable to grow in low serum (2% FBS), established Cl S1 mammary cells and SV40-transformed mammary epithelial cells replicated well. Cell densities achieved by the transformants were only slightly elevated in high serum (13% FBS) over normal cell values. All the transformants formed colonies on plastic and exhibited anchorage-independent growth in methylcellulose. Five of the transformed lines were tumorigenic in syngeneic animals, in marked contrast to the lack of transplantability usually observed with SV40-transformed mouse fibroblasts. Anchorage-independent growth was not a predictor of tumorigenic potential in this system. The transformants exhibited a spectrum of responsiveness to exogenous growth factors. This study establishes that the SV40-murine mammary cell system is a valid model for analyses of the process and consequences of epithelial cell transformation, in general, and mammary cell transformation in particular.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen

Mouse LTK- cells (H-2k) were transfected with a series of recombinant plasmids consisting of the herpes simplex virus type 1 thymidine kinase (TK) gene linked to fragments of SV40 DNA coding for portions of SV40 T antigen in pBR322, and TK+ transformants (LTK+) were selected in HAT medium. The TK+ transformants were analyzed for SV40 transplantation rejection antigen (TrAg) at the cell surface by reacting them with cytotoxic lymphocytes (CTL) generated to SV40 TrAg in C3H/HeJ (H-2k) mice. The results indicated that the cells transformed by pVBETK-1 and synthesizing full size SV40 large T antigen were efficiently lysed by SV40 CTL. In addition, cells transformed by the plasmid pVBt1TK-1 and synthesizing a truncated 33 K T antigen were also found to be susceptible to lysis by the CTL. However, LTK+ cells that were transformed with the plasmid pVBt2TK-1 and which synthesized a truncated T antigen of 12.3 K did not provide a target for SV40 CTL nor did pVBETK-1-transformed cells that did not express any of the SV40 tumor antigens. Only the pVBETK-1-transformed cells that express 94 K T antigen were able to immunize mice against a challenge of syngeneic SV40-transformed cells. These results suggest that the TrAg expression at the cell membranes of transformed cells may be associated with the proximal half of SV40 T antigen.     

Transformation of mammalian cells by DNA-containing viruses following photodynamic inactivation

Simian papovavirus 40 (SV40) and herpes simplex virus (HSV) types 1 and 2 were nactivated by exposure to fluorescent light after replication in cells pretreated with neutral red. Exposure of hamster embryo fibroblast cells to appropriately inactivated virus yielded clones of cells showing loss of contact inhibition. Cell lines were established from clones arising from “transformation” by all viruses tested. SV40 tumor (T) antigen was detected in the nuclei of the SV40 transformed cells and herpes simplex virus specific antigens were demonstrated in the cytoplasm of both type 1 and type 2 transformed cells. Virus was not recovered from the transformed cells. The results indicate that DNA-containing viruses are able to transform mammalian cells after photodynamic inactivation of infectivity.     

The small-t protein of SV40 is required for loss of actin cable networks and plasminogen activator synthesis in transformed rat cells

Of a variety of properties characteristic of SV40 transformed rat embryo fibroblast cells we find that those acquired by minimally transformed cells (the ability to grow clonally and in medium supplemented with low levels of serum) depend only on the large-T protein of SV40 while those properties associated exclusively with fully transformed, tumorigenic cells (growth suspended in methyl cellulose or agar, loss of cytoplasmic actin networks, and synthesis of plasminogen activator) are more likely to occur in cells transformed by viruses which encode the small-t protein as well. In addition one of these properties of the fully transformed cells, PA synthesis, is at least partially independent of large-T activity. Thus the phenotypic difference between the two classes of transformants may be related to the presence or absence of small-t influence within the cell.     

Losses of 3p, 11p, and 13q in EJ/ras-transformable simian virus 40-immortalized human uroepithelial cells.

Five independent clones of Simian virus 40 (SV40)-immortalized human uroepithelial cells (CK/SV-HUC) were established after transfection of HUC cultures from the same tissue donor with plasmids encoding SV40 large T and small t antigen genes. Each CK/SV-HUC clone contained a unique SV40 integration site, and all expressed similar levels of SV40 mRNA. All five clones were nontumorigenic, but clones 2, 4, and 5 tumorigenically transformed after transfection at P19 with mutant EJ/ras and also spontaneously after 40 serial passages in vitro. In contrast, CK/SV-HUC clones 1 and 3 did not transform when either approach was used. These differences in transformability among CK/SV-HUC clones could not be predicted based on differences in SV40 gene expression nor on any in vitro growth property tested. In cytogenetic analyses, a transformable clone showed losses of three chromosome arms containing putative cancer suppressor gene regions, including 3p14----pter, 13q, and 11p, whereas the nontransformable clones showed none of these losses. Thus these data indicate that genetic losses on 3p, 11p, and 13q may contribute to tumorigenic transformation of SV40-immortalized human uroepithelial cells.     

A comparison of human papovavirus T antigens.

A comparison was made of the T antigens induced in transformed cells or infected permissive cells by representatives of three categories of human papovavirus. The transformed hamster cell lines employed contained T antigen induced by either the BK or RF strains of papovavirus associated with human renal allografts; the JC strain of papovavirus from progressive multifocal leukoencephalopathy (PML), or a variant of SV40 virus isolated from PML. The human papovavirus T antigens were also compared with that of a human cell line transformed by SV40 of simian origin. Anti-T antibody prepared in hamsters against each of the hamster cell lines was absorbed with crude T antigen from each cell line, and the unabsorbed and absorbed antisera were tested for residual T antibody against each cell line, or against infected permissive cells by immunoperoxidase (IP) staining and complement-fixation (CF) tests. In unabsorbed antisera, T antibodies from each cell line cross-reacted with all T antigens in IP tests, and CF tests showed that T antisera reacted preferentially with T antigen induced by homologous virus. Absorpminants. T antigens of the two urine-derived strains, BK and RF, were identical or nearly so, but were clearly separable from T antigens of JC virus, PML-derived SV40 or simian-derived SV40. JC T antigen was intermediate, being more closely related to T antigens both of BK virus and SV40 virus than the latter were to each other. The T antigen of PML-derived SV40 could be distinguished from the T antigen of simian-derived SV40 and the T antigen of the SV40 variant from human brain was more closely related to those of the other human-derived papovaviruses than was the T antigen of SV40 from monkey kidney.     

Transformation by purified early genes of simian virus 40

A rapid soft-agar assay using baby hamster kidney (BHK21 cl.13) cells has been developed to establish the functional roles for the large T and small t antigens of SV40 in transformation. Plasmids expressing either large T or small t antigens of SV40 have also been constructed and these plasmids have been used separately or in combination for transformation. A large T clone, pD3-05, containing a deletion in the small t-specific coding region [0.584-0.54 map units (mu)], transformed a low-background subclone of baby hamster kidney (BHK21 cl.13) cell line and F111 rat fibroblasts to anchorage independence at a low level (10-20 and 1%, respectively, of an early region clone from wild type [WT], pW2). A WT-derived small t clone, pW2-t, containing a deletion in the large T-specific coding region (0.373-0.169 mu), did not transform F111 cells, but transformed BHK21 cells at a very low level (about 2% of pW2). Another WT-derived small t clone, pW2-t/B1, containing a larger deletion in the large T-specific coding region (0.512-0.169 mu), did not transform either BHK21 or F111 cells. However, cotransformation with pD3-05 clone and pW2-t or pW2-t/B1 clone increased the frequency of transformation to about the same level as that of pW2. The ability of the small t clones to enhance the transformation efficiency of the large T clone was not due to recombination between the two plasmids, since cotransformation with pD3-05 and a small t clone without the polyadenylation [poly(A)] signal sequence from WT, pW-t8, did not increase the frequency of transformation. When the frequency of transformation was determined by the focus assay using F111 cells, pD3-05 transformed as well as pW2. Also, cotransformation with pD3-05 and pW2-t/B1 did not increase the frequency of focus formation. Therefore, the small t antigen was not required for this morphological transformation.     

Variant lines of mouse kidney cells transformed by an SV40tsA mutant with growth properties of wild-type transformed cells at nonpermissive temperature.

MKSA207 cells, a BALB/c mouse kidney line transformed by a tsA mutant of SV40, are temperature-dependent for the expression of the 'standard transformed phenotype'. At the permissive temperature (33.5 degrees C), the mKSA207 cells resembled wild-type (wt) SV40 transformants; they contained the intranuclear SV40 T antigen, grew to high saturation density in monolayer culture in either 10% or 0.5% serum, and also in methylcellulose suspension culture and became multinucleate in cytochalasin B. At the nonpermissive temperature (39.8 degrees C), the mKSA207 cells lost some of their transformed properties; they grew only to low density in 10% serum, hardly grew at all in 0.5% serum or in methylcellulose suspension culture, and remained mono- or binucleate in cytochalasin B. At 40 degrees C in low serum, mKSA207 cells lost the intranuclear T antigen and when fed 10% serum at 39.8 degrees C, accumulated large amounts of T antigen in the cytoplasm. Derivatives of mKSA207 have been selected at 39.8 degrees C in liquid medium and methylcellulose suspension culture. The heat adapted lines, like wt SV40 transformants, exhibited the standard transformed phenotype at both 33.5 and 39.8 degrees C. It is unlikely that acquisition of temperature-independence for the transformed phenotype was due to reversion of the tsA gene to wild-type because the heat-adapted cell lines displayed the cytoplasmic T antigen at 39.8 degrees C, characteristic of the parental mKSA207 cells and SV40 rescued from one of the heat-adapted lines was temperature sensitive for growth. The T antigen levels (complement fixation units per 10(6) cells) of heat-adapted lines grown at 39.8 degrees C were comparable to those of mKSA207 cells grown at 33.5 or 39.8 degrees C.     

Rescue of defective SV40 from a transformed mouse 3T3 cell line: Selection of a specific defective

Small-plaque type SV40 yields various defectives containing deletions, insertions, and substitutions. To select a specific defective lacking a part of the late genes, we attempted to rescue a defective mutant from a transformed mouse cell line. A triple heterokaryon culture was prepared which consisted of monkey cells, 3T3 cells transformed by defective SV40, and 3T3 cells transformed by large-plaque type SV40 that yields no defective virus having the capacity to direct T antigen synthesis. Virus recovered from the heterokaryon culture was propagated in monkey cells, and defective light virions were concentrated by CsCl equilibrium centrifugation. The virions and their DNA thus obtained were found to direct T antigen synthesis but not synthesis of virion antigen. Heteroduplex DNA molecules presumably consisting of a complete and a defective strand had mostly two single-stranded loops. The rescued defective SV40 genome appeared to have a deletion and an insertion at two separate and fixed sites.     

Comparison of SV40 induced antigens on the surface of cultivated cells by a cytolytic microassay

By means of a direct cytolytic assay, SV40 specific antisera were shown to kill SV40 T antigen positive AL/N mouse cell lines, including lines which were repeatedly passed through the syngeneic mouse as tumors. The sera also killed a polyoma transformed SV40 T antigen negative AL/N cell line, but not untransformed or spontaneously transformed AL/N embryo cell lines. With the antisera tested there was also no lysis observed on SV40 T antigen positive Balb/c mouse, hamster or human cell lines, although by use of a competition assay SV40 specific common antigens were detectable on the surface of these cells.     

Biological activities of deletion mutants of simian virus 40.

Mutants of Simian virus 40 (SV40) with large deletions in the early or late regions of the genome were tested for biological activity. Deletion mutants lacking portions of both late genes (B/C and D), but with an intact early genomic segment, were able to induce T antigen in infected cells, replicate their DNA in the absence of helper virus, stimulate thymidine incorporation into cellular DNA, and transform mouse and hamster cells. Cells transformed by late deletion mutants were shown to contain the mutant genome by a fusion-complementation rescue procedure. Deletion mutants lacking substantial portions of the early genomic region, including those segments where tsA mutants map, lacked all of the above activities. However, both early and late deletion mutants interfered with SV40 DNA replication.     

An SV40 transformation revertant due to a host mutation: isolation and complementation analysis.

We have isolated an SV40 transformation revertant cell line, CL1L, by selection for normal cells whose growth is inhibited under low serum conditions. This line expresses a single, wild-type copy of large T antigen, yet is not transformed. It is not retransformable by transfection of SV40 DNA or infection with a recombinant retrovirus encoding large T antigen. Resistance to transformation therefore appears to be due to a cellular mutation. Fusion of CL1L cells to normal 3T3 cells or SV40-transformed cells results in somatic cell hybrids that are transformed, indicating that resistance is recessive. In addition, fusion of CL1L cells to another SV40 transformation-resistant line, A27, results in transformed hybrids, indicating the existence of discrete complementation groups with respect to SV40 transformation.     

Enhanced protein phosphorylation in SV40-transformed and -infected cells

We have studied the phosphorylation of cellular phosphoproteins and, in more detail, of SV40 T antigen and the cellular protein p53 in SV40 tsA-transformed cells. As detected by radiolabeling cold-sensitive tsA1499- or heat-sensitive tsA58-transformed rat fibroblasts with [32P]orthophosphate or by in vitro labeling extracts with [gamma-32P]ATP the hyperphosphorylation of certain cellular phosphoproteins including p53 and also of free SV40 large T antigen and T antigen complexed with p53 is strictly correlated with the expression of the transformed phenotype. This hyperphosphorylation can be observed as early as 30 min after shifting to the temperature where the cells expressed the transformed phenotype and, furthermore, it is dependent on protein synthesis. To evaluate the influence of a functional T antigen and to exclude properties of individual transformants we 32P labeled in vitro cellular proteins from rat F111, mouse NIH 3T3, and monkey TC-7 cells infected with tsA58 or tsA1499. In tsA58-infected cells we found a heat-sensitive enhancement of protein phosphorylation just as in tsA58 transformants. In tsA1499-infected monkey cells we observed a heat-sensitive and in abortively infected rat or mouse cells a cold-sensitive hyperphosphorylation of proteins. Thus in tsA-transformants and in various tsA-infected cells we found a strong correlation among the transformed phenotype, functions of T antigen, and the phosphorylation of various cellular proteins and in particular T antigen and p53.     

Fate and expression of simian virus 40 DNA after introduction into murine cells under nonselective conditions

When SV40 infects mouse cells, it does not replicate but instead causes neoplastic transformation of a small percentage of the cells. It is unknown, however, what happens to the virus in those cells that do not become transformed. We introduced SV40 into mouse cells by nonselective means, either by cotransfection of SV40 DNA with a selectable marker or by random cloning of SV40-infected cells. We analyzed the fate of viral DNA sequences, expression of T antigens, and transformation properties of these cells. We found that, upon infection, viral DNA integration occurs at a frequency that is at least 10-fold higher than the frequency of transformation. The majority of these cells are not transformed due to lack of expression of T antigen. One cell line which expresses a truncated T antigen is not transformed. We have mapped the viral sequences in the genome of these cells and find that integration in the large T intron is probably responsible for the defect. Lack of transformation can therefore be attributed to both cellular and viral factors, namely, introduction of viral DNA into cells that are resistant to transformation or integration of viral DNA in such a way that T antigen expression is prohibited.     

Effect of nuclear localization of large tumor antigen on growth potential of SV40-transformed cells

Transport-defective mutants of PARA, an SV40-adenovirus 7 hybrid virus, induce the synthesis of SV40 large T-antigen (T-ag) that is not transported to the nucleus and accumulates in the cytoplasm (cT-ag) of infected and transformed cells. The ability of cT mutants of PARA to induce phenotypic transformation of hamster embryo fibroblasts was examined. The cytoplasmic localization of T-ag was not stable, as extensive subculturing resulted in cell populations comprised of mixtures of cells which expressed T-ag in either the nucleus and/or the cytoplasm. Clonal analysis resulted in the selection of two cell populations: one cell type expressed T-ag solely in the cytoplasm and the second contained a mixture of cells which expressed T-ag in the nucleus and/or the cytoplasm. Serial subculture of clonal cell lines containing T-ag exclusively in the cytoplasm resulted again in the evolution of mixed populations which expressed T-ag in the nucleus and/or the cytoplasm. Analysis of synchronized cultures of such mixed clonal lines revealed that the intracellular distribution of T-ag was under the influence of the cell cycle; T-ag was present in the nucleus during S-phase, the period of maximal DNA synthesis, and in the cytoplasm during other phases of the cell cycle. These results suggest that a selective advantage exists during in vitro culturing for cells with a partial reversion of the cT phenotype. Examination of the growth properties of clonal lines in vitro indicated that cells in which T-ag could be detected in the cytoplasm, either continuously or during certain phases of the cell cycle, exhibited reduced growth potential under stringent culture conditions relative to wild-type transformants, behaving more like minimal transformants. In addition, the capacity for tumor induction in weanling hamsters by PARA(cT)-transformed hamster cells was reduced substantially in comparison to wild-type transformed cells. These results suggest that the constant presence of SV40 T-ag in the nucleus promotes maximal expression of the transformed cell phenotype.     

Differential requirement for SV40 early genes in immortalization and transformation of primary rat and human embryonic cells

A series of recombinant plasmids carrying various DNA fragments of SV40 early region were used to test for their ability to immortalize primary cultures of rat embryo (RE) and human embryonic kidney (HEK) cells. When primary RE cells were transfected with plasmids containing an entire early region of wild-type SV40- or a deletion mutant in the small tumor (t) antigen, dl 1410-DNA, they were all immortalized. The immortalized cells could grow in soft-agar medium and produced large tumor (T)-antigen. Cultured RE cells transfected with pW2-t, which contains a deletion in the large-T-specific coding region, also gave rise to continuous cell lines. Interestingly, two of nine RE lines immortalized by pW2-t could also grow in soft-agar medium. The plasmid pW-t8 carrying a similar fragment of SV40 DNA as pW2-t, but lacking the processing and polyadenylation signal sequences, also immortalized RE cells. Surprisingly, the plasmid pD-t1 which contains neither the intact large-T nor the small-t function also immortalized RE cells. However, the RE lines immortalized by pW-t8 or pD-t1 were unable to grow in soft-agar medium and displayed a wide range of growth phenotypes. On the contrary, when primary HEK cells were used for immortalization experiments, only those SV40 plasmids carrying the intact large-T function were able to generate immortalized lines. The growth properties of these immortalized HEK lines can be categorized into two groups. Those HEK lines immortalized by the large T alone grew slightly denser and rounder than their parental normal HEK cells, while those immortalized by both the large-T and small-t antigens grew extremely fast, reached higher density, piled up on each other, and were anchorage independent. In addition, when these SV40 plasmids were used to directly transform primary HEK cells by the focus assay, the large-T clone, pD3-05, only transformed HEK cells to form light foci. Transfection by the large-T plus the small-t sequences either in cis or in trans, did increase the frequency of focus formation, and gave rise to dense foci which could grow in soft-agar medium.     

Cell lines derived from tumors induced in syngeneic rats by FR 3T3 SV40 transformants no longer synthesize the early viral proteins

Several SV40-transformed FR 3T3 rat cell lines formed tumors upon inoculation to syngeneic immunocompetent Fisher rats. These tumors, which appeared only after a long latency period, showed a fast rate of growth. Tumor-derived (TD) cell lines were established in culture from several tumors induced by independent transformants, and their properties were analyzed. Though TD cells were highly tumorigenic, their level of transformation in culture was similar to that of the original transformants. They did not synthesize detectable amounts of the two early viral gene products, the large-T and small-T polypeptides. However, the transformation-associated cellular p53 protein was detected in all of them by [35S]methionine labeling and immune precipitation with monoclonal antibodies directed against the mouse p53. Growth in the animal apparently counterselected the cells expressing the early viral proteins, and hence, possibly, the tumor-specific transplantation antigen. This selection was mediated at least in part by the T-cell immune response, as the tumors induced by the same transformants in nu/nu mice still expressed the nuclear T-antigen. Absence of expression of the early viral region was frequently correlated with the loss of the integrated SV40 DNA. Some tumors, however, still contained early viral DNA sequences, which were, in even fewer cases, transcribed into RNA. These results altogether suggest that tumor formation by the FR 3T3-SV40 transformed cells in immunocompetent rats requires two events, the selection for the acquisition of a high tumorigenic potential, and against the expression of the early viral genes. Only the first of these two events was observed upon tumor formation in nude mice.     

An SV40 mutant oncoprotein has a nuclear location

T147 is an SV40 mutant that makes a normal small t antigen and a large T antigen that is only 147 amino acids long. We have introduced a second mutation into the genome of T147 which eliminates its ability to encode small t antigen. We show that this double mutant is able to transform C3H10T1/2 mouse cells in a focus assay and F111 rat cells in an agar suspension assay, demonstrating that the transforming domain of T antigen is located within its amino-terminal 147 amino acids. We also show that the T147 mutant T antigen, like wild-type T antigen, has a nuclear location. However, in contrast to wild-type T antigen, which is also found in the plasma membranes of wild-type transformed cells, we fail to detect any mutant T antigen associated with the plasma membranes of T147 transformants.