Expression and thermal stability of simian virus 40 tumor-specific transplantation antigen and tumor antigen in wild type- and tsA mutant-transformed cells.

We have explored aspects of a suggested relationship between the expression of simian virus 40 tumor-specific transplantation antigen (TSTA) and tumor antigen (TA). A unique rat embryo cell line transformed by a temperature-sensitive A mutant that loses TA during exposure to the nonpermissive temperature (A28-RE) was found to lose TSTA. On the other hand, a typical control tsA-transformed cell line (A239-MB) expressed both TA and TSTA at the non-permissive temperature. TA in lysates obtained from A239-MB cells was found to be three to four times more thermolabile by covwt-mb) when incubated at either 33 or 40 degrees C. These data complement previous reports using TA from lytic infection and are consistent with the suggestion that TA is virus encoded. In contrast to TA, which even in wild-type-transformed cells was completely destroyed in less than 10 min at 50 degrees C, TSTA, assayed in vivo by tumor rejection, and tumor-specific surface antigen(s) TSSA) defined by an in vitro cytolytic assay, were thermostabile. Even after 24 h of incubation of extracts of 50 degrees C, high levels of TSTA and TSSA activity were present. Since these surface antigens when obtained from cells transformed by tsA mutants were also thermostabile, they could not be distinguished from the wild-type antigens. These results (i) indicate a coordinate expression of TA and TSTA in transformed cells; (ii) confirm that TA is virus encoded; and (iii) confirm that tha antigenic and immunogenic determinants that characterize TA and TSTA activities are distinct. However, the possibility that TSTA, like TA, is of viral rather than cellular origin is not excluded.     

Demonstration of multiple antigenic sites of the SV40 transplantation rejection antigen by using cytotoxic T lymphocyte clones

Multiple antigenic sites on the simian virus 40 (SV40) tumor-specific transplantation antigen (TSTA) were detected by the use of cytotoxic T lymphocyte (CTL) clones isolated from continuous cultures of SV40-specific CTL (H-2b). Two independently derived clones, K11 and K19, specific for the SV40 TSTA in association with H-2Db, each recognized a different antigenic determinant of the SV40 TSTA. This conclusion was based on the observation that a human papovavirus BK virus (BKV) transformed cell line, which possesses a T antigen serologically cross-reactive with that of SV40, was lysed by a heterogeneous population of SV40-immune lymphocytes and by clone K19 but not by K11. Therefore, these CTL clones must recognize two different antigenic determinants of the SV40 TSTA:K19 recognizes a cross-reactive determinant of the SV40 and BKV TSTA, whereas K11 is reactive against an SV40-specific determinant.     

Insensitivity of a ricin-resistant mutant of Chinese hamster ovary cells to fusion induced by Newcastle disease virus.

The simian virus 40 (sv40) tumor antigen (T-antigen) and tumor-specific transplantation antigen (TSTA) have been partially purified and studied to clarify their relationship. The T-antigen and the TSTA were partially purified from nuclei of SV AL/N cells, and SV40-transformed mouse embryo fibroblast line, by precipitation with ammonium sulfate and chromatography on DEAE- and DNA-cellulose. The T-antigen was assayed by complement fixation, and the TSTA was assayed by its ability to immunize mice against SV40-containing ascites tumor cells. When T-antigen- and TSTA-containing preparations were sedimented through sucrose gradients, each antigen had a major peak of activity at a sedimentation coefficient of 6.7 and minor peaks in other regions. Antiserum against T-antigen (from tumor-bearing hamsters) immunoprecipitated the TSTA activity. A preparation of T-antigen from human SV80 cells, which exhibited only one protein band after sodium dodecylsulfate-polyacrylamide gel electrophoresis, had TSTA activity when as little as 0.6 microgram of protein per mouse was used for immunization. These experiments demonstrate that the T-antigen, the product of the SV40 early A gene is capable of inducing specific immunity against transplantation of SV40-transformed tumor cells in mice.     

Cell surface antigens coded for by the human chromosome 7

Human-mouse somatic cell hybrids, containing chromosome 7 from an SV40-transformed human cell line as the only human chromosome, were injected into the same inbred strain of mouse as the mouse parental cell, and the humoral immune response assayed. A cell-surface antigen(s) coded for by the chromosome 7 common to all human fibroblastic cell lines tested and also found on African green monkey kidney cell lines was demonstrated. No reactivity to SV40-induced TSTA was detected.Abbreviations used in this paper are as follows SV40 simian virus 40 - MEM minimal essential medium - FBS fetal bovine serum - FITC fluorescein isothiocyanate - C121 53-87 (1) clone 21 - Cl36 53-87-3 clone 36 - 52-62 52-62 (1) clone 5 subclone 9 - MSV murine sarcoma virus - T tumor - TSTA tumor-specific transplantation antigen - RIA radioimmunoassay - PBS phosphate-buffered saline - PBS⁺ PBS with sodium azide and FBS - IgG immunoglobulin - cpm counts per minute     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.     

Primary in vitro sensitization of murine lymphocytes against isogeneic and allogeneic cells transformed by simian virus 40.

Primary in vitro sensitization of murine lymphocytes to isogeneic and allogeneic cells transformed by simian virus 40 (SV40) is described. The results of specificity studies utilizing cytotoxic effector lymphocytes obtained by in vitro immunization indicate that SV40 transformation results in the expression of tumor-specific antigens which are recognized by cytotoxic effector cells. Moreover, the studies demonstrate that expression of tumor-specific antigens on transformed cells is associated with a reduction in the functional expression of normal histocompatibility antigens.     

Flat revertants of temperature-insensitive transformants induced by simian virus 40 tsA mutants lose their ability to express T-antigen.

Temperature-insensitive transformants that contained simian virus 40 sequences at only one or a few sites in the rat chromosome and that were induced by a temperature-sensitive A gene mutant of simian virus 40 were used to select flat revertants (revertants that had lost the transformed phenotype). The isolation was performed at the nonpermissive temperature so as not to select against temperature-sensitive transformants. Nonetheless, all of the revertants examined had lost their ability to express the T-antigen at both temperatures, and all contained rearrangements of the integrated simian virus 40 sequences. These results are most compatible with the hypothesis that the T-antigen of simian virus 40 is required for the maintenance of the transformed state even in temperature-insensitive cell lines.     

Generation of cytotoxic lymphocytes by SV40-induced antigens

In order to study the correlation of in vivo tumor transplantation immunity and in vitro immunologic assays, cell-mediated cytotoxicity against SV40-transformed cells was studied in AL/N strain mice by using 51Cr-release assay. Killing of SV40-transformed AL/N fibroblast cells was observed by spleen cells of AL/N mice immunized with syngeneic SV40-transformed cells. Immunization with the solubilized SV40 tumor-specific transplantation antigen (TSTA) that induced transplantation immunity in vivo did not elicit cytotoxic spleen cells in vitro. However, the spleen cells from mice immunized with solubilized TSTA and then sensitized in vitro with SV40-transformed cells became cytotoxic against SV40-transformed fibroblasts. Similarly, SV40 TSTA (T antigen) purified by immunoprecipitation was able to prime the lymphocytes in AL/N mice: the primed lymphocytes could differentiate into cytotoxic lymphocytes upon in vitro stimulation by SV40-transformed cells. These data indicate that SV40 TSTA (T antigen) plays a role in the induction of cytotoxic lymphocytes.     

Characterization of tau antigens isolated from uninfected and simian virus 40-infected monkey cells and papovavirus-transformed cells.

Tau antigens (also known as cellular or nonviral tumor antigens) were detected in uninfected and simian virus 40-infected monkey cells after immunoprecipitation with serum from hamsters bearing simian virus 40-induced tumours (anti-T serum). These two proteins (56,000 daltons) were digested to similarly sized peptides with various amounts of Staphylococcus aureus V8 protease. The Tau antigen isolated from infected monkey cells was closely related but was not identical to the corresponding protein from human cells transformed by simian virus 40, as determined by two-dimensional mapping of their methionine-labeled tryptic peptides. Hamster cells transformed by various primate papovaviruses (simian virus 40, BK virus, and JC virus) synthesized indistinguishable Tau antigens, as determined by two-dimensional peptide mapping. When tested by the same procedure, these proteins and the ones made in monkey and human cells were found to be related to the Tau antigens isolated from simian virus 40-transformed mouse and rat cells. Based on these results, an "evolutionary tree" was constructed to show the relationship among the methionine-containing tryptic peptides of all of these proteins.     

Somatic cell hybrids between mouse peritoneal macrophages and SV40-transformed human cells. III. Identification of surface antigens coded for by human chromosomes 7 and 17.

Somatic cell hybrids between SV40-transformed human cell lines and mouse peritoneal macrophages (MPM) containing either human chromosome 7 or 17 carrying the SV40 genome were injected into mice syngeneic to the mouse parental cells. Since either chromosome 7 or 17 was the only human chromosome present in the hybrids used as immunogens, the humoral immune response to gene products coded for by either chromosome was assayed. Using a sensitive radioimmunoassay, we were able to identify noncross-reactive cell-surface antigen(s) specifically coded for by either human chromosome 7 or 17, and present in normal, tumor-derived and virus-transformed human cells. However, no reactivity against SV40 tumor-specific surface antigen (TSSA) could be detected in the antisera.     

Density dependent inhibition of both growth and T-antigen expression in revertants isolated from simian virus 40-transformed mouse SVT2 cells.

Phenotypic revertants were isolated from simian virus 40-transformed cells in order to examine the relationship between simian virus 40 T-antigen expression and G1 arrest of growth. Revertant clones with increased adherence were selected from cultures of SVT2, a simian virus 40-transformed BALB/c mouse cell line, and screened to find arrestable revertant clones which inhibited DNA synthesis when crowded. The clones selected from untreated SVT2 were unstable and showed little or no inhibition of DNA synthesis when crowded. Stable revertants were found after treatment of SVT2 with Colcemid to increase ploidy. The stable revertants all lost most transformed growth properties tested, including tumorigenicity, but only a few showed the same degree of inhibition of DNA synthesis at high cell density as BALB/3T3. All revertant clones expressed T antigen at low cell density. Three revertants showed coordinate inhibition of DNA synthesis and apparent loss of T antigen at high cell density. We suggest that changes in gene dosage rather than mutations caused the altered properties of the new revertants and that continued DNA synthesis in confluent cultures may be the transformed phenotype that requires the least simian virus 40 T antigen.     

Subcellular Localization of Simian Virus 40 Large Tumor Antigen

The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [(35)S]-methionine- or (32)P(i)-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of approximately 96,000 was detected in both subcellular fractions. Minor components of approximately 68,000 and approximately 56,000 with anti-T reactivity which labeled with [(35)S]methionine were also detected in both fractions from H-50 cells, as were components of approximately 140,000 and approximately 56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in (32)P(i)-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [(3)H]thymidine labeling, NADH-diaphorase activity, and Na(+)-K(+)-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization, and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen.     

Relationship between simian virus 40 large tumor antigen expression and tumor formation in transgenic mice.

A line of transgenic mice containing the simian virus 40 (SV40) large tumor antigen gene under the control of the viral enhancer-promoter expressed this viral protein in the brains of these mice within the first 2 weeks after birth. Multiple foci of anaplastic cells formed in the choroid plexuses of these mice at 36 to 41 days after birth, and normal tissue coexisted with these transformed foci. Immunoperoxidase staining to detect the SV40 T antigen showed tumor-specific expression of nuclear T antigen at late times in tumor development, approximately 90 to 100 days and thereafter. The level of SV40 T antigen, on a per cell basis, appeared to be lower in the great majority of choroid plexus cells at earlier times in tumor development. These results suggest that low levels of tumor antigen (14 to 36 days) are present before detectable pathology (36 to 41 days) and the level of T antigen per cell is higher in rapidly growing late-stage tumors (older than 90 days).     

Stabilization of the 53,000-dalton nonviral tumor antigen is not required for transformation by simian virus 40.

We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent about one-half of the coding region for large T antigen. We present evidence here that F8dl is able to transform mouse cells in a focus assay and that cell lines derived from these foci exhibit fully transformed phenotypes, have integrated mutant genomes, and express mutant-encoded proteins. This result implies that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for the maintenance of transformation. In addition, we have found that cells fully transformed by F8dl produce a 53,000-dalton nonviral tumor antigen (p53) that is as unstable as the p53 of untransformed cells. From this result we infer that transformation by simian virus 40 does not require the stabilization of p53.     

Detergent solubilized and molecular weight estimation of tumor specific surface antigen from SV40 virus transformed cells.

Mild detergent treatment of SV40 transformed mouse embryo fibroblasts, followed by (NH₄)₂SO₄ precipitation of solubilized proteins and rate zonal centrifugation in a sucrose gradient, provided direct and rapid determination of the sedimentation coefficient (4S) of tumor specific surface antigen(s) (TSSA), as detected by an antibody dependent, cell lysis assay. A molecular weight range of 50,000–60,000 was estimated for the TSSA. These results agree with those also obtained from Sephadex G-150 exclusion chromatography.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses VIII. Association of Simian Virus 40 Transplantation Antigen with a Specific Region of the Early Viral Genome

Two of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrids induce SV40 transplantation resistance in immunized hamsters. These two hybrids, Ad2(+)ND(2) and Ad2(+)ND(4), contain 32 and 43% of the SV40 genome, respectively. The pattern of induction of SV40 transplantation antigen (TSTA) by the various hybrids differentiates TSTA from both SV40 U and T antigens. Since the SV40 RNA induced by both these hybrids is early SV40 RNA, these findings confirm that TSTA is an early SV40 function. By combining available data on SV40 antigen induction by these hybrids with electron microscopy heteroduplex mapping studies, the DNA segment responsible for the induction of SV40 TSTA can be inferred to lie in the region between 0.17 and 0.43 SV40 units from the site on the SV40 chromosome cleaved by E. coli R(1) restriction endonuclease.     

Relationship Among Tau Antigens Isolated from Various Lines of Simian Virus 40-Transformed Cells

In addition to the virus-specified tumor antigens, simian virus 40-transformed cells contain at least one other protein which can be immunoprecipitated with serum from animals bearing simian virus 40-induced tumors. This protein, which is designated Tau antigen, has an apparent molecular weight of 56,000 as determined by electrophoresis on acrylamide gels. The relationship among Tau antigens isolated from different lines of simian virus 40-transformed cells was examined by comparing the methionine-labeled tryptic peptides of these proteins by two-dimensional fingerprinting on thin-layer cellulose plates. In this fashion, we initially determined that the Tau antigens isolated from three different lines of transformed mouse cells were very similar. Second, we found that Tau antigen isolated from a line of rat transformants was closely related, but not identical, to the mouse cell Tau antigens. Approximately 70% of their methionine peptides comigrated in two dimensions. Finally, we showed that Tau antigen isolated from a line of transformed human cells was only partially related to the mouse and rat proteins. About 40% of the methionine peptides of the human protein were also contained in the Tau antigens from the other two species. These results strongly indicate that the Tau antigens isolated from these various simian virus 40-transformed cell lines contain common amino acid sequences.     

Phosphorylation of T-antigen and control T-antigen expression in cells transformed by wild-type and tsA mutants of simian virus 40.

Chinese hamster lung (CHL) cells transformed by wild-type simian virus 40 (cell line CHLWT15) or transformed by the simian virus 40 mutants tsA30 (cell lines CHLA30L1 and CHLA30L2) or tsA239 (cell line CHLA239L1) were used to determine the rates of turnover and synthesis of the T-antigen protein and the rate of turnover of the phosphate group(s) attached to the T-antigen at both the permissive and restrictive temperatures. The phosphate group turned over several times within the lifetime of the protein to which it was attached, with the exception of the phosphate group in the tsA transformants at 40 degrees C, which turned over at the same rate as the T-antigen protein. The steady-state levels of the T-antigens (molecular weights, 92,000 [92K] and 17K) and the amount of simian virus 40-specific RNA was also determined in each of the lines. The CHLA30L1 line contained two to three times more early simian virus 40 RNA than the CHLA30L2 line; although neither line formed colonies in agar at 40 degrees C, CHLA30L1 overgrew a normal monolayer at 40 degrees C. The rate of 92K-T-antigen synthesis was 1.5 times faster in CHLA30L1 than in CHLA30L2 at 33 degrees C and 4 times faster at 40 degrees C. The different phenotype of these two presumably isogenic cell lines seem to be related to the levels of the T-antigens. The ratios of the 92K T-antigen to the 17K T-antigens were similar in the two lines. Transformed CHL cell lines, unlike transformed mouse 3T3 cell lines, were found to contain very small amounts of the 56K T-antigen.     

A fragment of the simian virus 40 early genome can induce tumors in nude mice.

Cell lines transformed by simian virus 40 mutant F8dl (deleted from 0.168 to 0.424 map units, corresponding to the carboxy-terminal 62% of the wild-type simian virus 40 large tumor antigen) are tumorigenic in nude mice. Four of five C3H10T1/2 cell lines transformed by F8dl were tumorigenic in nude mice, whereas two of two wild-type transformants were tumorigenic.     

Simian virus 40 gene A function and maintenance of transformation.

Transformants have been isolated after infection of rat embryo cells at 33 C with either wild-type simian virus 40 or with the temperature-sensitive gene A mutants, tsA7 and tsA28. Examination of properties usually associated with transformation such as growth in 1% serum, growth rate, saturation density, and morphology show that these properties are temperature dependent in the tsA transformants characterized, but are not temperature dependent in the wild-type transformants that have been examined. In the most thoroughly characterized tsA transformants the expression of T antigen also appears to be temperature dependent. These data suggest that an active A function is required for the maintenance of transformation in these cells. In the lytic cycle, the A function is involved in the initiation of DNA synthesis. Thus transformation by simian virus 40 may be the direct consequence of the introduction of the simian virus 40 replicon and the presence of its DNA initiator function, which causes the cell to express a transformed phenotype.