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.     

Biologic properties of viable deletion mutants of simian virus 40 (SV40) rescued from the cells of an SV40-induced hamster lymphocytic leukemia

A lymphocytic leukemia induced by the oncogenic DNA simian virus 40 (SV40) in an inbred LSH/SsLak Syrian golden hamster was evoked to produce infectious SV40 by fusion of the leukemia cells with grivet monkey kidney (GMK) cells and by exposure of the leukemia cells to the chemical inducers mitomycin C and cycloheximide. Plaque-purified viable substrains of the rescued SV40 when studied by restriction endonuclease digestion of viral DNA were found to contain small deletions within the Hind III restriction fragment C. These deletions lay near the viral origin of DNA replication. Ten plaque-purified substrains of the rescued virus identified by immunofluorescence as being SV40 were found, when compared to the wild-type SV40, to replicate slowly and to form small plaques. Although these substrains transformed NIH/3T3 cells as efficiently as the wild-type SV40 in tissue culture, they were generally less oncogenic in vivo--7 of the 10 failed to induce tumors. The 3 oncogenic SV40-rescued substrains were not found to exhibit "lymphocytotropism," i.e., the capacity to infect and neoplastically transform preferentially hamster lymphocytes. Thus the hamster lymphocytic leukemia originally induced by the wild-type SV40 was most likely a chance-stochastic event rather than the result of tropism-determinism mediated by the virus, as is usually the case with leukemogenic RNA viruses.     

Transformation of rat hepatocytes by transfection with simian virus 40 DNA to yield proliferating differentiated cells

Cultured hepatocytes from adult Fischer 344 rats were transformed by virion or cloned simian virus 40 (SV40) DNA using the calcium phosphate method. Transformation by SV40 occurred in either serum-supplemented medium or chemically defined medium (CDM). The frequency was greatest in serum-supplemented medium but transformants did not remain differentiated. In contrast, SV40 transformants developed less frequently in CDM, but retained differentiated functions. The frequency of transformation was enhanced by treatments that stimulated cell proliferation, in particular supplementing CDM with epidermal growth factor. Hepatocytes transformed in CDM were epithelial in morphology, secreted albumin, transferrin, hemopexin, and expressed the enzyme glucose-6-phosphatase, all characteristics of normal liver. Transformants did not produce detectable levels of alpha-fetoprotein, a marker of fetal or abnormal liver. We conclude that (a) hepatocytes can be transformed by transfection with SV40 DNA; (b) the frequency of transformation is enhanced by stimulating DNA synthesis; and (c) the transformed cells retain specific functions of normal hepatocytes in situ. Using this system it will be possible to study transformation of hepatocytes by viral and cellular oncogenes and to determine their effects on hepatocellular differentiation.     

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.     

Simian virus 40 (SV40) production from SV40-transformed human amnion cells of established lines.

Sixteen established cell lines of simian virus 40 (SV40)-transformed human amnion cells were examined for SV40 production. Many of these lines produced SV40 for extensive periods. Virus production had not ceased for 2 lines after 18 months, for 3 lines after 12 months, and for 3 lines at 3 months after recovery from "crisis". Three lines became virus-free in the first month, 1 line in the second month, 1 in the third month, and 1 in the fourth month, and 2 lines stopped virus production between 6 and 11 months after recovery. The virus titers were relatively low. Inclusion body-containing cells were infrequent. In contrast, in most cultures of SV40-transformed human fibroblasts rescued from crisis, no infectious virus was demonstrated, although exceptions have been reported. Virus was produced after heterokaryon formation of cells of the virus-free amnion lines with CV-1 cells in the presence of inactivated Sendai virus, as observed for SV40-transformed human fibroblasts. During the crisis period, some of the SV40-transformed amnion cells produced substantial amounts of virus. Titers decreased during the later periods of crisis. The most pronounced decrease in titers was in cultures from which established lines were recovered.     

Correlation of collagen synthesis and procollagen messenger RNA levels with transformation in rat embryo fibroblasts

A line of normal rat embryo fibroblasts was transformed with N-methyl-N'-nitro-N-nitrosoguanidine (a chemical carcinogen), SV40 and polyoma virus (two DNA viruses), and Rous sarcoma virus (an RNA tumor virus). In this study, we report a comparison of the levels of collagen synthesis and procollagen messenger RNA (mRNA) in 13 lines selected after transformation with one of these agents. Collagen synthesis and procollagen mRNA levels were compared with the degree of transformation determined from morphology, saturation density, growth in agarose, and tumorigenicity in nude mice. Each class of transformants had a characteristic level of collagen synthesis; this level correlated inversely with the degree of transformation of the rat embryo fibroblasts. In N-methyl-N'-nitro-N-nitrosoguanidine and SV40 transformants which were moderately transformed, collagen synthesis was hardly affected, but, in polyoma virus and Rous sarcoma virus transformants which were more severely transformed, collagen synthesis was 30 to 48% and 12 to 25%, respectively, of control levels. Type I procollagen mRNA activity measured in RNA from nine of the lines by an in vitro translation assay also decreased with increasing severity of transformation. Procollagen mRNA levels were reduced to about one-half of control levels in one SV40 transformant and to 17 to 23% of controls in polyoma virus and Rous sarcoma virus transformants. We conclude that, in this series of rat fibroblast lines, transformation with different agents resulted in characteristic levels of collagen synthesis and that collagen synthesis was most reduced in the cells which were most transformed by other criteria.     

Reversibility of the transformed and neoplastic phenotype. IV. Effects of long-term interferon treatment of C3H/10T1/2 cells transformed by methylcholanthrene and SV40 virus

The effects of long-term treatment with mouse interferon on the phenotype of untransformed C3H/10T1/2 cells and cloned derivatives transformed by methylcholanthrene (MCA) and Simian virus 40 (SV40) were investigated. Continuous presence of interferon induced morphologic reversion, with the development of thick, submembranous filaments in MCA-transformed cells, whereas no morphological effects were detected in SV40-transformed cells. Interferon inhibited the proliferation of all three cell lines and maintained low saturation densities. However, prolonged treatment of MCA-transformed cells with interferon rendered them tolerant toward the anti-proliferative and antiviral activities of interferon although 2-A synthetase activity was induced. Interferon treatment reduced the capacity of both MCA and SV40-transformed cells to form colonies in agar and decreased the tumorigenicity of MCA-transformed but not SV40-transformed cells in mice. These results indicate that the same cell type transformed by different means has different sensitivities to a number of interferon-induced changes in the cell phenotype.     

New antigens in SV40 transformed cells. I. Demonstration of three new antigenic constituents in several cell lines of different species transformed by the SV40

New antigens in SV40 transformed cells. I. Demonstration of three new antigenic constituents in several cell lines of different species transformed by the SV40 The soluble components of different cell lines in various species (hamster, mouse, rat and dog) were compared by means of precipitation test in agar. Some lines were spontaneously transformed, others were transformed by SV40 virus and oncogenic viruses. The utilization of rabbit and hamster antisera, specific for a clone of SV40 transformed cells (TSV₅Cl₂) demonstrated the presence of three new antigens in the various cell strains transformed by SV40. These antigens are different from the nuclear T antigen and probably also from the transplantation antigen located on the cell membrane.     

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.     

Demonstration of oncogenic potential of mammalian cells transformed by DNA-containing viruses following photodynamic inactivation.

The oncogenic properties of hamster embryo cells transformed by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and SV40 virus following photodynamic inactivation using neutral red were determined by subcutaneous inoculation into newborn Syrian hamsters. Cells transformed by all three viruses produced palpable tumors after different latent periods. Histopathological examination showed that HSV-2 tumors were fibrosarcomas and metastases were often seen in the lungs. HSV-2 primary tumors were reinoculated subcutaneously into weanling hamsters; they developed palpable tumors within 2 weeks. HSV-specific antigens were detected in the cytoplasm and/or on the surface of both the HSV-1 and HSV-2 tumor-cell cultures by the indirect immunofluorescence technique. The same method revealed SV40 tumor antigen in the nuclei of the SV40 tumor cells. Sera from HSV or SV40 tumor-bearing hamsters gave positive reactions when tested against HSV-infected hamster cells or SV40-infected monkey cells, respectively. These results demonstrate that herpes simplex virus and SV40, whose infectivity was lost following photodynamic inactivation, retained the virus genetic information necessary for transformation of normal cells to an oncogenic phenotype.     

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.     

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.     

Expression of SV40 T antigen during the cell cycle of sv40-transformed cells.

The expression of the nuclear SV40-induced T antigen was measured by microfluorimetry on individual, asynchronously growing SV40-transformed cells which had been stained with hamster T-antiserum by the indirect immunofluorescence method. The same individual cells were first measured for T antigen and then for DNA by Feulgen microspectrophotometry. A linear correlation was observed between the two parameters. T antigen expression was also measured in cell populations arrested at different phases of the cell cycle. Results of both types of experiments show that the expression of the gene (s) for T antigen in transformed cells increases during DNA replication and reaches its highest level in G2 nuclei. During mitosis T antigen is found in the cytoplasm.     

The transformation of BHK 21 hamster cells by simian virus 40

Although BHK 21 cells remain refractory to direct transformation by SV40 virus even at multiplicities of infection as high as 10⁴ PFU per cell, transformed derivatives may be produced by cocultivation of the untransformed cells with monkey cells infected with SV40 virus. The characteristics of a cloned SV40-transformed line (C13/SV) have been studied. The cells showed several of the properties of BHK 21 cells transformed by other oncogenic viruses. High-passage variants of the line (C13/SV-M) metastasized from the primary tumour that they induced in hamsters. Metastatic foci were the result of dissemination of the cells, and not due to the release of infectious SV40 virus from implanted cells. Comparisons between the in vitro and in vivo growth properties of C13/SV and C13/SV-M cells did not reveal a correlation with metastasizing ability. The C13/SV-M cells also retained those properties of transformed cells that are probably associated with surface alterations, although the SV40-specific transplantation antigen may be partially absent or masked in these cells.     

Chromosome complement and SV40 transformation of cells from patients susceptible to malignant disease.

A comparative study has been made of fibroblasts obtained from patients with differing susceptibilities to malignant disease, both with respect to their chromosome complements and their transformation with SV40 virus. Fibroblasts from 2 Bloom's syndrome patients were found not to have raised SV40 transformation rates and no correlation was found between chromosome abnormality per se and transformation. Of 2 cell types with greatly increased rates, one was derived from a neurofibromatosis patient and the other from an A-T heterozygote. When SV40 DNA was employed as the transforming agent for the latter, the transformation rate was no longer raised.     

Detection of SV40 T antigen genome in human gliomas

The monkey polyomavirus simian virus 40 (SV40) has been reported to be associated with tumorigenesis of human neoplasms, mainly brain tumors. However, it remains controversial whether the virus really exists in human neoplasms and how the virus transforms human cells in vivo. We investigated the presence of SV40 T antigen genome in 33 human glioma tissue specimens from Japanese patients with different histopathologies by means of the polymerase chain reaction (PCR) followed by Southern blotting. The SV40 T antigen genome was amplified in 4 of the 13 ependymomas (31%) and 3 of the 20 other histotypes of gliomas (15%), whereas in the 22 nontumoral brain tissue specimens, only one case was found to be positive. DNA sequencing confirmed the PCR products to be those of SV40 T antigen. The findings thus suggest that the SV40 genome appears to exist in a certain population of brain tumors from Japanese patients, and that it may also play a role in the oncogenicity or maintenance of the transformed state.     

Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences.

The normal genomic stability of human cells is reversed during neoplastic transformation. The SV40 large T antigen alters the DNA content in human endometrial stromal cells in a manner that relates to neoplastic progression. Human endometrial stromal cells were transfected with a plasmid containing the A209 temperature-sensitive mutant of SV40 (tsSV40), which is also defective in the viral origin of replication. Ninety-seven clonal transfectants from seven different primary cell strains were isolated. Initial analysis revealed that 20% of the clonal populations (19/97) had an apparent diploid DNA content, 35% (34/97) had an apparent tetraploid DNA content, and the remainder were mixed populations of diploid and tetraploid cells. No aneuploid populations were observed. Diploid tsSV40 transformed cells always give rise to a population of cells with a tetraploid DNA content when continuously cultured at the permissive temperature. The doubling of DNA content can be vastly accelerated by the sudden reintroduction of large T antigen activity following a shift from non-permissive to permissive temperature. Tetraploid tsSV40 transfected cells have a lower capacity for anchorage-independent growth and earlier entry into 'crisis' than diploid cells. These results indicate that during the pre-crisis, extended lifespan phase of growth, the SV40 large T antigen causes a doubling of DNA content. This apparent doubling of DNA content does not confer growth advantage during the extended lifespan that precedes 'crisis'.     

Limitations and utility of a cytolytic assay for measuring simian virus 40-induced cell surface antigens.

Antisera were produced against an SV40-transformed cell line in the syngeneic AL/N mouse. With a microcytolytic assay, the specificity of antisera produced by various immunization schedules and their ability to lyse numerous SV40-transformed cell lines were determined. Various AL/N mouse cell lines, newly transformed by SV40 and cloned, were found to be lysed by the antisera. When tumors were induced by SV40-transformed cells in the syngeneic mouse and cell lines were reestablished from tumors and such procedures were repeated, the susceptibility to serum-mediated cytolysis of the sublines was the same as that of the original SV40-transformed cell line, in spite of differences in tumorigenicity. Polyoma virus-transformed AL/N cell lines were also lysed while AL/N embryo cells, untransformed by SV40 or by polyoma, were not. SV40-transformed T-antigen-positive BALB/c mouse or hamster cell lines or a T-antigen-positive tissue cultured human cell were also resistant to lysis. A competition type of microassay demonstrated specific inhibition of the serum-mediated cytolysis by all of the SV40 T-antigen-positive cell lines tested. Thus, the lack of lysis of cells did not necessarily indicate the absence of SV40-induced surface antigens. The polyoma-transformed AL/N cell line also inhibited the antisera, but to a lesser extent, suggesting the possibility that SV40 and polyoma virus transformation may result in the appearance of partially common cell surface antigens.     

Rat cell line 3y1 and its virogenic polyoma- and sv40- transformed derivatives.

Cell lines were established from cultures derived from Fischer rat embryos according to the transfer schedule described by Todaro and Green (1963) for mouse 3T3 cells where cell crowding and serum exhaustion were kept to a minimum. Cell growth rate did not decline greatly during the course of successive 3-day transfers. Like 3T3 cells the rat cell lines possess very low saturation desities under standard culture conditions. A clonal cell line with a relatively high plating efficiency as obtained from one of the cell lines, 3YL. In these cloned cultures, virus growth was not detectable upon infection with SV40, while a small amount of virus was produced upon infection with polyoma virus. Morphological transformation of the cloned 3Y1 cells by SV40 and polyoma virus could be assayed with single-hit kinetics and with effieiencies comparable to those of the previously available transformation systems for each virus. Independent cell lines transformed by SV40 were consistently virus-free and all the lines tested produced SV40 upon fusion with permissive monkey cells. Most of the independent transfromed cell lines isolated after polyoma infection appeared to be virus-free, although the cultures of some lines produced a small amount of polyoma virus spontaneously after a prolonged cultivation. Most of the virus-free polyoma-transformed lines produced virus upon fusion with permissive mouse cells.