New antigens in cells transformed by the SV40 virus. II. Evidence of their cytoplasmic localization in a cell line (TSV5 CL2) of hamster fibroblasts transformed by SV40

A clone of hamster fibroblasts transformed by SV40 virus (TSV₅ CL₂) induced antibodies in hamsters against new antigenic constituents not present in the untransformed fibroblasts. The antibodies in the immune sera against histocompatibility antigens and those against nuclear T antigen were absorbed. The absorbed sera contained antibodies which agglutinated the transformed cells. The sera contained also precipitating antibodies against new antigens present in a crude extract of TSV₅ CL₂. These antibodies were directed to common new antigens present in SV40-transformed fibroblasts from different species. Immunofluorescence and the “indirect enzyme-labelled antibody technique” were used for the cellular localization of the antigens corresponding to the precipitating antibodies. Both techniques showed that these antigens were localized in the cytoplasm, mostly in the microsome-ribosomal fractions, much less on the cell membrane and not at all in the nucleus.     

Properties of transformed hamster cells containing SV40 tumor antigen in the cytoplasm

The properties of hamster cells containing SV40 tumor (T) antigen in the cytoplasm, rather than the nucleus, were determined. Eight cell lines were established from eight tumors induced by hamster embryo fibroblasts transformed in vitro by PARA (2cT)-adenovirus 7. Six cell lines contained only cytoplasmic SV40 T-positive cells while two were a mixture of nuclear T-positive and cytoplasmic T-positive cells. All the cell lines contained SV40 S antigen, all caused the production of SV40 T antibody in vivo, and four elicited the production of adenovirus T antibody. The cell lines seem to have acquired an infinite life span in vitro. The localization of T antigen apparently can be a stable phenomenon because five of the cell lines have retained T antigen exclusively in the cytoplasm for over 40 passages in tissue culture. The cytoplasmic T antigen could be detected by complement fixation in addition to immunofluorescence. Cytoplasmic T-positive cells were readily transplantable in vivo and contained SV40 TSTA demonstrable by both immunogenicity and immunosensitivity procedures. Two of the three cytoplasmic variants of PARA appear to be weakly oncogenic in newborn hamsters.     

Differences in the manifestation of virus-specific surface antigen between cells transformed by SV40 and UV-irradiated SV40

Marked differences in the manifestation of SV40-specific surface (S-) antigen were found between hamster cell lines transformed in vitro and in vivo by SV40 and UV-irradiated or photodynamically inactivated SV40. In the mixed hemagglutination reaction (MHA) cell lines induced by non-irradiated SV40 yielded positive results when tested against specific antisera up to dilutions of 1:1280. In comparison, cell lines induced by inactivated SV40 only showed positive reactions in the MHA-test in dilutions up to 1:80 or 1:160 respectively. The demonstration of small quantities of SV40-specific S-antigen in cell lines transformed by inactivated SV40 could only be achieved by use of hyperimmune sera produced by a special immunization procedure. However, if these cells were tested in the MHA reaction against anti-S-sera, produced according to conventional methods described in the literature, they yielded negative results. All cell lines with diminished quantities of demonstrable S-antigen caused malignant tumors when inoculated into adult hamsters. The capacity of UV-irradiated SV40 to induce a specific transplantation resistance in adult Syrian hamsters was investigated. The irradiated virus induced a markedly reduced transplantation resistance.     

Incorporation of SV40-tumor specific transplantation antigen (SV40-TSTA) in vesicular stomatitis virus grown in SV40 transformed hamster cells (author's transl)

Highly purified and inactivated VSV (VSV/TSV-5 C1.2) grown in SV40 transformed hamster cells, TSV-5 C1.2, induces in hamster a transplantation immunity against host-tumor cells. Immunization with VSV/TSV-5 C1.2 gave more than a 10-fold protection against TSV-5 C1.2 tumor cells. The transplantation immunity confered by the immunization is specific for tumor cells which possess SV40-TSTA. VSV/TSV-5 C1.2 protects hamsters against another SV40-transformed cell line, EHSVi-C1.1. In contrast, immunization with VSV/EHB, a purified preparation of VSV grown in spontaneously transformed hamster cells, EHB, gave no protection to TSV-5 C1.2 cell challenge. It was concluded that the presence of SV40-TSTA activity in VSV/TSV-5 C1.2 virions was due to incorporation of this tumor cell antigen in the viral envelope. The existence of cell antigens in VSV/TSV-5 C1.2 and VSV/EHB preparations was also shown in vitro by Cr⁵¹ release assay. The anti-VSV/TSV-5 C1.2 rabbit serum reacted specifically with SV40-transformed cells, TSV-5 C1.2 and EHSVi-C1.1.     

The SV40 S antigen; A carcinoembryonic-type antigen of the hamster?

In addition to hamster tumor cells induced or transformed by SV40, hamster anti-SV40 S (Surface) antiserum also reacted with non-SV40-exposed cell lines spontaneously induced (BHK 21) or transformed by heterologous oncogenic DNA and RNA viruses in the indirect membrane fluorescent antibody test. The antiserum titered equally with both BHK and SV40-transformed cells and the reaction could be absorbed with either of these cell lines, or with hamster embryo. The antiserum also reacted with early hamster embryo cells, although various organ cells from late fetuses, newborns and adults were negative. Mouse cell lines spontaneously induced (BALB/c-3T3) or transformed by SV40 and adenovirus 12 were also non-reactive. The results suggest that the antigen in question is a hamster somatic antigen present during embryonic life and derepressed in lines of actively growing cells.     

Differences in the capacity of simian virus 40 (SV40) tumor antigens on cells, membranes and in soluble form to induce transplantation immunity in hamsters and mice.

The immunogenicity of the SV40 tumor-specific transplantation antigen (TSTA) on cells, cell particulates and solubilized membranes was studied in mice and in Syrian hamsters. Immunizations were done with various concentrations of tissue-culture-passaged, non-virus-releasing transformed cells, purified cell membranes and in some cases purified nuclei and papain-solubilized membranes obtained from several species, including the mouse, hamster, man, and sheep. All transformed cell lines were T-antigen-positive. The immunosensitive mKSA line of BALB/c mice and the immunosensitive SV34 cell line of the hamster were used for tumor challenge. All materials, regardless of source and of type of preparation, were strikingly immunogenic in the mouse but only SV40 virus and SV34 (hamster) cells provided protection against tumor cell challenge in the hamster. Also, in a limited study, BKV-transformed hamster cells and purified cell membranes and JCV-transformed hamster cells were found to be immunogenic by the tumor rejection assay in the mouse but not in the hamster. SV40 immunization did not protect the hamster against BKV- and JCV-transformed hamster cells. These results are discussed in terms of possible different specificities resident on the TSTA molecule.     

In vivo induction of tumor-specific immunity by glycolipid extracts of SV40-transformed cells

Glycolipid extracts were prepared from various Syrian golden hamster cell lines, either SV40-transformed or spontaneously transformed. To detect possible SV40-TSTA activity of the glycolipid preparations, normal hamsters were inoculated with different glycolipid extracts and were subsequently challenged with an SV40 tumor-cell line. Significant immunoprotection against SV40 tumor challenge was induced with glycolipids obtained from SV40-transformed cell lines. This was expressed as complete tumor rejection or as a decrease in tumor growth rate, when compared to controls. No protective effects were induced with glycolipid extracts from spontaneously transformed cells. Results suggest that tumor-specific glycolipids synthesized in cells transformed by SV40 virus could act as tumor transplantation antigens responsible for specific tumor rejection in syngeneic hosts.     

New antigens in cells transformed by the SV40 virus. 3. Presence of an SV40-coded antigen on the surface of different cell lines transformed by this virus (author's transl)

The sera of hamsters carrying tumours induced by injection of TSV₅Cl₂ cells and the sera of animals immunized with these cells or with SV40 itself or its purified capsids have a slow cytotoxicity activity in the presence of fresh guinea pig complement on SV40-transformed cell strains of any origin. The responsible antigen is dependent on the SV40 genome and is associated with the cytoplasmic membranes of the transformed cells. By means of absorption of the antisera with subcellular fractions it has been correlated with antigen “C” present in the cytoplasm of SV40-transformed cells. It is different from antigen “S” shown by the immunofluorescence test, and its relation with TSTA is discussed.     

Characterization of the surface proteins of SV40-transformed mouse and human cells: absence of SV40-specific proteins

The proteins of a number of SV40- and spontaneously transformed mouse and human cell lines were compared in an effort to identify a surface protein which would correspond to the SV40 tumor-specific transplantation antigen (TSTA). Analysis of the one- and two-dimensional electrophoretic patterns of 35S-methionine-labelled total proteins and 125I-labelled surface proteins of several of these cell lines failed to reveal the presence of proteins specific to transformation by SV40. Antisera were prepared against SV40- and spontaneously transformed mouse cells in syngeneic mice. In serological assays, these antisera reacted with surface antigens common to both SV40- and spontaneously transformed mouse cell lines. Electrophoretic analysis of the 125I-surface-labelled proteins which these antisera immunoprecipitated from extracts of SV40- and spontaneously transformed mouse and human cells identified a set of common surface proteins with apparent molecular weights of 15, 46, 50, 72, 77, 105, 150 and 230kdal. No SV40-specific surface proteins were detected. Two of the transformed cell surface proteins (105 and 150kdal) were present as well in membrane fractions of 35S-methionine-labelled primary mouse kidney cultures. The proteins of the primary cultures could not be iodinated by lactoperoxidase suggesting that these proteins were present at a "cryptic" location at the surface of normal cells. We were not able to obtain serological or immunochemical evidence for the presence of SV40 large T-antigen at the surface of any of the SV40-transformed cell lines tested using either hamster anti-SV40 tumor sera, a rabbit antiserum against SDS-denatured gel-purified large T-antigen or antisera against SV40-transformed mouse cells. In conjunction with the report that large T-antigen released from disrupted SV40-transformed cells will bind to cell surfaces (Lange-Mutschler and Henning, 1982), we consider the possibility that the specific rejection of SV40-induced tumors by sensitized animals is the result of immunological reactions against both common transformation-related surface antigens and SV40 T-antigen from disrupted cells that has bound to the surface of other tumor cells.     

Specific glycolipid antigen(s) in SV40-transformed cell membranes

A glycolipid extract was prepared from an SV40-transformed hamster cell line (EH-SV) according to the Folch partition procedure. The glycolipids from the aqueous layer were incorporated in liposomal membranes composed of lecithin/sphingomyelin/cholesterol (1:1:2 by weight). This liposomal preparation was inoculated in Syrian hamsters to raise immune sera. The sera were absorbed with trypsinized "normal" hamster cells (EH-N) and tested on various cell lines by the indirect immunofluorescence technique. When used for staining living cells, the immune serum produced a distinct cell-surface fluorescence with SV40-transformed cell lines regardless of the cell origin (e.g., rat or hamster). No reaction was observed with heterologous Py-transformed cell lines, spontaneously transformed cells, or sera from non-immunized hamsters. When used for staining acetone-fixed cells, the antiglycolipid serum reacted specifically with a thermostable antigen in the nuclear envelope and the cytoplasm of SV40-transformed cells. The sera lack interfering SV40 T reactivity. The results indicate the presence of related SV40-specific glycolipid antigen(s) in the plasma membrane, the nuclear membrane and probably other endomembranes of SV40-transformed cells.     

Transformation of rat and guinea-pig cells in vitro by SV40, and the transplantability of the transformed cells

Cell cultures of rat and guinea-pig kidneys inoculated with simian virus 40 (SV40) were found to undergo morphological changes characteristic for SV40 transformation. Cell lines of rapidly-growing transformed rat cells were obtained and found to be free from infectious SV40. They contained a specific antigen which was demonstrated in complement fixation tests with serum from hamsters bearing SV40 tumors. When the transformed rat cells were injected subcutaneously into the autologous hosts, tumors histologically classified as sarcomas developed in animals which had been pretreated by X-ray irradiation. Tumor cells from one of the rats were passaged in vivo and gave rise to sarcomas of high malignancy also in non-treated animals. A tumor line was thus established in rats. It contained the specific complement-fixing “tumor” antigen but no infectious SV40. Transformed guinea-pig cells autotransplanted into the irradiated host caused a small tumor which regressed within a few weeks. The results indicate that autotransplantation of in vitro- transformed cells into irradiated animals is a more promising way of obtaining SV40 tumors in different animals than the inoculation of newborns with virus.     

SV40 tumor rejection induced by vesicular stomatitis virus bearing SV40 tumor-specific transplantation antigen (SV40-TSTA). I. Specificity of immunoprotection and effect of enzyme treatment on TSTA activity.

Highly purified vesicular stomatitis virus (VSV) was obtained from VSV-infected SV40-transformed hamster cell lines. Immunization with this virus protected hamsters against challenge with SV40-transformed cells (TSV5-cl2). This protection was obtained regardless of the source of the SV40-transformed cells (e.g. cat, rat, hamster) used to produce VSV, and was therefore associated with the SV40 tumor-specific transplantation antigen (SV40-TSTA). Furthermore, when grown on spontaneously transformed cell lines or on cells transformed by a different oncogenic DNA virus, such as polyoma virus, the VSV failed to protect against the SV40-induced tumor. It was concluded that the SV40-TSTA activity of purified VSV is due to the incorporation of SV40-TSTA within the viral envelope. When VSV was treated with proteolytic enzymes (bromelain, trypsin) no loss of TSTA-induced tumor rejection was observed, although VSV had lost its ability to induce virus-neutralizing antibody. This clearly demonstrates that the TSTA activity is not related to the viral spikes. Phospholipase C suppressed the TSTA activity but neutralizing activity was still detectable in the anti-VSV sera. The results presented here demonstrate that the protection afforded by VSV is highly specific. It is particularly interesting that SV40-TSTA activity may be conveyed by the lipid core of the viral envelope.     

SV40 tumor rejection induced by vesicular stomatitis virus bearing SV40 tumor-specific transplantation antigen (SV40-TSTA). II. Association of SV40-TSTA activity with liposomes containing VSV glycolipids.

Highly purified vesicular stomatitis virus (VSV) was obtained from VSV-infected SV40-transformed and from "normal" hamster cell lines. A glycolipid extract was prepared from these VSV preparations according to the Folch partition procedure. These glycolipids were rendered immunogenic to the Syrian hamsters when incorporated within liposomal membranes composed of lecithin/sphingomyelin/cholesterol (1/1/2 by weight). When the glycolipids were extracted from VSV grown on cell lines (TSV5-cl2 and EHSVi-cl1) which contained the SV40 tumor-specific transplantation antigen (SV40-TSTA), it was possible either to induce a tumor rejection or at least to slow the growth of the tumor in Syrian hamsters challenged with TSV5-cl2 cells. No protection was obtained in animals treated with liposomes containing glycolipids extracted from purified VSV grown on SV40-TSTA-negative cells (EHB). The SV40-TSTA could be a glycolipid of the transformed cell membrane which is incorporated within the VSV envelope.     

HSV- and chemical carcinogen-induced amplification of SV40 DNA sequences in transformed cells is cell-line-dependent

Eleven simian virus 40-transformed cell lines from 5 different species were tested for their ability to amplify integrated simian virus 40 DNA upon infection with herpes simplex virus type I or treatment with various chemical carcinogens. Four cell lines were positive only for virus-induced gene amplification and two lines were positive for both carcinogen- and virus-induced gene amplification. Individual cell lines were assayed for the presence of an intact SV40 origin of replication, the expression of a functional SV40 T-antigen, and permissivity to herpes simplex virus replication. These parameters were found to be positive in all 6 amplification-competent cell lines. The ability of herpes simplex virus to amplify SV40 DNA sequences in transformed cells is greater than that of chemical carcinogens and can be suppressed by specific inhibitors of the herpes virus-encoded DNA polymerase.     

Interspecies-, species- and type-specific T antigenic determinants of human papovaviruses (JC and BK) and of Simian virus 40.

Immunofluorescence tests, absorption studies and quantitative analysis by a very sensitive 51Cr microcomplement fixation (CF) technique were used to define the degree of relatedness between the tumor (T) antigens induced by human papovaviruses, strain JC and BK, with simian virus 40(SV40) and mouse polyoma virus (PyV). Antisera against JCV, BKV, SV40 and PyV T were raised in tumor-bearing hamsters. The data obtained indicate that T antigens of JCV, BKV and SV40 possess various subspecificities which can be distinguished and looked upon as interspecies-, species- and type-specific antigenic determinants. It was found that JCV T and BKV T synthesized in transformed hamster cells share about the same amount (20%) of interspecies cross-reacting antigen with SV40 T from H-50 cell extracts (transformed hamster cells). Although hamster cells transformed by PyV showed definite PyV T reactivity, no cross-reactivity, at least with the sera used, was found with human papovavirus and SV40 T antigens. Furthermore, degree of heterogeneity was observed within the T antigen complex derived from different SV40-transformed cells.     

Detection of specific surface antigens by colony inhibition in cells transformed by papovavirus SV40

Specific antigens at the surface of SV40-transformed hamster cells were demonstrated by the in vitro colony inhibition and immunofluorescence tests. Antisera were prepared by inoculating hamsters either with purified SV40 or with human or marmoset SV40-transformed cells. The inhibition of colony formation by the transformed cells ranged from 46 to 100% in the presence of specific antibody and complement. Nontransformed hamster cells were not inhibited by the immune sera. Of the 22 sera tested, 12 were positive in the colony inhibition test and 14 were positive in the immunofluorescence test. Eleven of the 22 sera tested were positive in both tests. This positive correlation between the colony inhibition and immunofluorescence tests suggests that the two tests are detecting the same or similar antigens.     

Papovavirus SV40: similarity of tumor antigens in transformed virus-free hamster cells and in virusinfected monkey cells

Sera from hamsters bearing tumors induced by papovavirus SV40 reacted with tumor antigen prepared either from cells transformed by, or from cells infected with, the homologous virus. Serum titurs against both antogens were comparable. A high degree if correlation was obtained with the use of either complement-fixation or immunofluorescence to detect the antigen. The immunoflurescent intranuclear antigen present in cells transformed by SV40, an the early virus-induced intranuclear antigen found during the cytolytic cycle following infection of monkey cells by SV40, therefore appear to be similar, and they are either identival with or similar to the antigens detected by complement-fixation techniques. Synthesis of antibody against this antigen was detected only in hamsters bearing tumors induced by SV40 or by cells transformed by the virus. Hamsters immunized with SV40 usually reject cells transformed by the homologous virus; the few vaccinated animals which fail to reject such cells also fail to produce antibodies against tumor antigen although the tumor cells in such animals continue to synthesize the antigen.     

Alterations of the thymic selection process in transgenic mice expressing SV40 large T antigen

We generated SV40 T antigen transgenic mice (lines SVT125, SVT127, and SVT248) which developed unique thymic carcinomas originating from thymic cortical epithelial cells. In these mice we observed alterations in the thymic selection process not reported before in SV40 T antigen transgenic mice. Along with tumor cell growth, thymocytes increased in number and the proportion of CD4 or CD8 single positive cells rose to 10 times the normal level. Expression of SV40 T antigen was detectable by Northern analysis in thymic stromal cells but not in thymocytes. Thymic stromal cell lines, derived from the thymic tumor, produced high levels of cytokines which caused morphological transformation and growth stimulation in hematopoietic stem cells, including fetal liver cells and bone marrow cells. These observations suggest that the unusual multiplication of thymocytes and the alterations in thymic selection are the result of the activity of thymic stromal cells transformed by SV40 T antigen. The cell lines derived from the tumor can thus be used to study cytokines involved in thymic differentiation of T cells. © 1996 Wiley-Liss, Inc.     

Expression of fetal antigens and tumor-specific antigens in SV40-transformed cells. I. Serological analysis of the antigenic specificities

The fetal antigens expressed in a variety of tumors have previously been shown to be different from tumor-specific antigens. The present study on the expression of fetal antigens in various SV40-transformed cells showed that more than one fetal antigen may be found in these cell lines, and that the expression of fetal antigens was not directly related to the function of the SV40 genome. These fetal antigens were not found in syngeneic spleen cells but were found in the normal spleens of some other strains of mice.