SV40-transformed cells express SV40 T antigen-related antigens on the cell surface

SV40 tumor antigen (T-Ag)-related antigens were detected serologically on the surface (surface T) of living SV40-transformed human and mouse monolayer cells by an ¹²⁵I-protein A binding assay. In immunofluorescence analysis, these cells were negative for surface T. However, on mKSA, a SV40-transformed mouse cell line grown in suspension or on SV40-transformed human and mouse monolayer cells put into suspension, surface T could be visualized by immunofluorescence microscopy. The antisera used in these experiments were raised in rabbits with purified, SDS-denatured SV40 T-Ag or came from hamsters bearing SV40 tumors. Both types of antisera had in common high titers against SV40 T-Ag (?1:1000). All these antisera were negative on normal cells or on polyoma virus-transformed cells. The specificity of both antisera for SV40 T-Ag-related binding sites on the surface of SV40-transformed cells were demonstrated by an ¹²⁵I-IgG blocking assay in which preincubation of the cells with rabbit anti-T-Ag serum inhibited the binding of hamster SV40 tumor serum to the cell surface by about 85%. These results demonstrate the expression of T-Ag-related antigens on the surface of living cells and, therefore, support the hypothesis that SV40 T-Ag-related antigens participate in the formation of the SV40-specific tumor transplantation antigen (TSTA).     

Association of SV40 large tumor antigen and cellular proteins on the surface of SV40-transformed mouse cells

A cellular protein with a molecular weight of about 53,000 (53K) and histocompatibility antigens (mouse H-2 antigens) have been reported to be associated with viral-specified proteins in transformed cells. We investigated whether such associations could be detected on the surface of SV40-transformed mouse cells. A differential immunoprecipitation technique was adapted so that surface-associated antigens could be detected independently from intracellular antigens. Cells grown as monolayers were enzymatically labeled with ¹²⁵I-Na using a lactoperoxidase-catalyzed reaction, or metabolically labeled with either [³⁵S]methionine or ³²P_i, and were then incubated with antisera against mouse H-2 antigens or SV40 large T-antigen (T-ag) or with monoclonal antibodies against mouse 53K nonviral T-antigen (nvT-ag). The cells were then disrupted with an NP40 solution, the extracts were clarified by centrifugation, and the immune complexes in the supernatant fluids adsorbed with protein A-containing Staphylococcus aureus. Internal antigens, present in the cell lysates, were precipitated by a second incubation with antiserum and the antigen-antibody complexes collected again with immunoadsorbent. The precipitated proteins were eluted and analyzed by SDS-polyacrylamide gel electrophoresis. Reconstruction experiments established that T-ag released from the nucleus during the extraction procedure was not combining with free antigen-binding sites on antibodies bound to the cell surface in the external reaction, that nuclear unbound T-ag was not exchanging with bound surface antigen during extraction, and that the surface reaction was not due to nuclear T-ag released from dead cells and nonspecifically adsorbed onto the surface of living cells. Iodinated 94K T-ag was specifically immunoprecipitated by T antibody during the external reaction; an iodinated 53K polypeptide was coprecipitated. Conversely, labeled T-ag and 53K were coprecipitated from surface-iodinated transformed cells by monoclonal antibodies against mouse 53K nvT-ag. Thus, it appears that SV40 large T-ag and cellular 53K protein are associated on the surface as well as within SV40-transformed mouse cells. In contrast, no detergent-stable complex between T-ag and mouse H-2 antigens was detected on the transformed cells. The possibility that molecular interactions between viral- and cell-coded proteins could be involved in determining some of the observed transformation-related cellular phenotypic changes is discussed.     

Comparison of SV 40 RNA from infected cells and SV 40 cRNA made by RNA polymerase from Eseheriehia coli

Summary SV 40 RNA from infected AGMK cells and SV 40 cRNA made by RNA polymerase fromE. coli on supercoiled SV 40 DNA are transcribed both largely asymmetrically but mainly from opposite strands. This was found by comparing both RNAs through competition hybridization and annealing experiments. 60–70% of the SV 40 RNA in the cell is transcribed from the opposite strand and 30–40% from the same strand as the SV 40 cRNA made by RNA polymerase fromE coli.     

Cell-surface antigens induced by avian RNA tumor viruses: detection by a cytotoxic microassay

A microcytotoxicity test for chicken embryo cells was introduced to search for new antigens after the infection of these cells with avian leukosis and sarcoma virus strains. At least two kinds of antigens could be demonstrated: (i) a subgroup-specific, presumably viral envelope (Ve) antigen on the surface of all avian tumor virus-infected chicken cells and (ii) a group-specific tumor-specific surface antigen (TSSA) common to all avian sarcoma virus transformed chicken cells regardless of the virus subgroup. A strong anti-TSSA response could also be elicited in chickens by the injection of either an avian leukosis virus strain or a nonconverting mutant of an avian sarcoma virus. In this assay system, the cytotoxic effect acting via the TSSA was considerably stronger than the one acting via the Ve antigen alone.     

SV40 T-antigen-related surface antigen: correlated expression with nuclear T-antigen in cells transformed by an SV40 A-gene mutant

Rat embryo cells transformed by the SV40 A-gene mutant tsA 28.3 were analyzed at permissive and nonpermissive temperatures for the expression of SV40 nuclear T-antigen (T-Ag) and of SV40 T-Ag-related surface antigen (“surface T”) by indirect immunofluorescence microscopy. At permissive temperature both antigens were detected with rabbit antiserum prepared against purified SDS-denatured T-Ag. Upon shift of tsA 28.3 cells to the nonpermissive temperature, expression of both nuclear T-Ag and of surface T decreased simultaneously. When the cells were returned to the permissive temperature, both nuclear T-Ag and surface T reappeared. Analysis of extracts from tsA 28.3 cells grown and labeled with [³⁵S]methionine either at permissive or at nonpermissive temperature by immunoprecipitation and SDS-polyacrylamide gel electrophoresis demonstrated that tsA 28.3 cells synthesized the T-Ag polypeptide only at the permissive temperature. The T-Ag polypeptide was not detected in extracts of cells grown and labeled at nonpermissive temperature. These results demonstrate a strong correlation between the expression of nuclear T-Ag, surface T, and the synthesis of the T-Ag polypeptide, suggesting that both nuclear T-Ag and surface T are products of the SV40 A-gene.     

SV 40 induced polypeptides in infected and transformed cells

5 structural (VP) and 3 non-structural (NSVP) SV40 induced polypeptides which display two kinetic patterns of synthesis are identified in infected cells. NSVP 1 and 2 are "early" functions of which only NSVP 1 is present in transformed cells; NSVP 3 is "late" function which is also present in a line (T-22) of transformed AGMK.     

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.     

Transformation of canine kidney cells by SV40

Canine kidney cells infected with SV40 virus led to the development of a transformed cell line (DKSV40). The transformed cells contained the nuclear T antigen of SV40 and grew in a disoriented pattern to a high saturation density. However, at 40°C the cells exhibited density-dependent inhibition of growth.     

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.     

Identification and biochemical analysis of DNA replication-defective large T antigens from SV40-transformed cells

Nine commonly studied Simian virus 40 (SV40)-transformed rodent cell lines were screened for tumor (T) antigens defective in SV40 DNA replication using a simple polyethylene glycol-mediated cell fusion assay. Each line contained a functional origin of SV40 DNA replication, as shown by fusion with Cos 1 cells. Fusion with uninfected monkey cells revealed that T antigens from two lines lacked detectable replicative activity, while T antigens from five other lines exhibited only very weak replicative activity. One line, and a tumor cell line derived from it, expressed T antigen with wild-type replication activity. Biochemical analysis of these proteins revealed defects in DNA binding activity and ATPase activity. One line expressed large T antigen defective in both activities. All of the lines contained complexes of T antigen with the cellular protein p53 and all of the T antigens exhibited nucleotide-binding activity. The results indicate that some of these lines may constitute a useful source of new replication-defective T antigens.     

Indomethacin-sensitive suppressor cells regulate the cell-mediated cytotoxic response to SV 40-induced tumor-associated antigens in mice

Specific secondary cytotoxic reactivity (as measured by ⁵¹Cr-release assay) against SV 40-induced tumor-associated antigens was generated in vitro in spleen cells of tumor-free (BALB/c × C57 BL/6)F₁ (CBF₁) mice immunized against a syngeneic SV 40-induced tumor of BALB/c origin (mKSA), following in vitro sensitization for 5 days with the relevant antigens in mixed lymphocyte-tumor cell cultures. In contrast, spleen cells of CBF₁ mice bearing the SV 40-induced tumor demonstrated suppressed specific secondary cytotoxic reactivity following incubation with the corresponding antigens. Spleens from tumor-bearing mice contained 4 times the number of mononuclear cells and 3 times the percentage of macrophages, as compared to spleens of normal mice. The percentage of B cells was also elevated in spleens of tumor-bearing mice. There was a slight reduction in the percentage of T cells. The cytotoxic reactivity of spleen cells of tumor-bearing mice was restored following removal of macrophages by either rayon adherence columns or iron and magnet, or incubation on plastic petri dishes. No such effect was seen with spleen cells of tumor-free or normal mice. Spleen cells of tumor-bearing mice inhibited the in vitro generation of secondary cytotoxic reactivity of spleen cells of tumor-free mice, sensitized in vitro with SV 40-induced tumor cells by mixing experiments. The suppressor cells were found to be macrophages by the 3 techniques for removal of macrophages described above. The addition of indomethacin (1–10 μg/ml), a noncompetitive irreversible prostaglandin synthesis inhibitor, to cultures of responding spleen cells from tumor-bearing mice and stimulating SV 40-induced tumor cells resulted in marked augmentation of spleen cells responsiveness. With higher indomethacin concentrations (100 μg/ml), no enhancement was seen. The augmenting effect was noted only when indomethacin was present during the initial 24 h of the 5-day culture. Indomethacin at 1–10 μg/ml had no effect on cytotoxic reactivity of spleen cells of tumor-free mice, whereas at higher concentrations (100 μg/ml) it had a strong suppressive effect. Preincubation of spleen cells of tumor-bearing mice with indomethacin for 3 days abrogated their ability to suppress the generation of secondary cytotoxic reactivity of spleen cells of tumor-free mice. It is hypothesized that indomethacin-sensitive suppressor macrophages regulate the immune responsiveness in tumor-bearing mice.     

Quantitative analysis of the immunogenic activity of SV40 virus and of tumor cells induced by this virus

A study of specific antitumor immunity created in Syrian hamsters by oncogenic virus SV40 and by tumor cells induced by the same virus showed that the level of specific resistance depends on the immunizing dose of virus and cells. Investigation of the resistance-inducing activity of a wild strain of SV40 virus showed that the minimal dose inducing resistance in hamsters was ten times higher than for the tsA-30 mutant of the virus. The minimal resistance-inducing dose of irradiated cells of a tumor induced by the same strain of SV40 virus was 9·10⁵ cells; a tenfold increase in the dose led to a significant increase in the level of specific antitumor immunity.Laboratory of Immunology of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 8, pp. 223–225, August, 1978.     

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.