SV40-transformed Cells Research Articles

Autoimmune expression of a cytoplasmic protein p60 in mice bearing metastasizing SV40‐transformed tumors

In STU mice bearing metastasizing SV40-transformed 51A-232B-M tumors, an immune response against a cellular 60kDa protein (p60) developed in about 50% of the tumor-bearing animals, in addition to the response against SV40 large T-antigen and cellular protein p53. The anti-p60 auto-immune response could be observed as early as 11 days after tumor challenge and was strictly linked with metastatic spread but was not a prerequisite for metastasis. Anti-p60 antibodies could not be detected in sera of animals bearing metastasizing Rous-sarcoma virus-transformed or methylcholanthrene-induced tumors, or in sera from human cancer patients with clinically confirmed metastatic spread. The anti-p60 auto-antibodies showed a broad cross-reactivity against components of similar size in a great number of cell lines of various species and in normal mouse tissue. The p60 auto-antigen is a cytoplasmic protein which is neither phosphorylated nor glycosylated in vivo. Immunoblotting performed with fresh cell lysates under non-reducing conditions using tumor-bearer sera revealed a diffuse p60 double band, but under reducing conditions only one sharp p60 band was observed. The reaction of p60 with anti-p60 auto-antibodies could be completely blocked by pre-treatment of fresh cell lysates with N-ethylmaleimide or p-chloromercuriphenyl sulfonate, or by oxidation in air prior to immunoblotting, indicating that the anti-p60 autoimmune response was directed against an epitope sensitive to SH-group-blocking reagents. Immunofluorescence studies with tumor-bearer sera showed only a very weak cytoplasmic fluorescence, possibly due to the nature of the p60 SH-groups in situ being masked. Immunoprecipitates with monoclonal antibodies against SV40 large T-antigen and p53 obtained from fresh cell lysates of SV40-transformed tumor cells contained no associated p60 auto-antigen. The p60 auto-antigen was purified from tumor cell homogenates with an enrichment factor of about 2,000; its iso-electric point is at pH 6.8. Determination of the biological half-life of p60 yielded a value of about 28 hr. The p60 auto-antibodies in pools of tumor-bearer sera taken at day 40 after tumor challenge all belonged to the IgG1 subclass.

Frequency Analysis of Simian Virus 40-specific Cytotoxic T Lymphocyte Precursors in the High Responder C57BL/6 Mouse Strain

Studies in this laboratory have shown that long term simian virus 40 (SV40)-specific cytolytic T lymphocyte (CTL) cultures established from the spleens of high responder C57BL/6 (B6; H-2b) mice exhibit a preference for the selection of H-2Db-restricted CTL clones. In this study, we have investigated the basis for this selection. Limiting dilution cultures were established using responder cells from the popliteal lymph nodes and the spleens of B6 mice immunized subcutaneously in the hind footpads or via the intraperitoneal route, respectively, with syngeneic SV40-transformed cells expressing a full length (1 to 708 amino acid residues) SV40 large T antigen. The relative frequency of CTL precursors (CTLp) able to expand in vitro in the presence of SV40-transformed stimulator cells and interleukin 2 and exhibit lytic activity against H-2b cells expressing full length T antigen ranged from 1/1900 to 1/15,000 in the popliteal lymph node and from 1/8000 to 1/55,000 in the spleen. In these two experimental systems, CTLp restricted to H-2Kb were apparently present at higher frequency than H-2Db-restricted CTLp. Furthermore, CTLp recognizing determinants within the amino-terminal or carboxy-terminal halves of T antigen were generated in approximately equal numbers. The relative affinity of SV40-specific CTL, assessed by inhibition with anti-Lyt 2 monoclonal antibody, indicated that CTL restricted to H-2Db interacted with their target with greater affinity than CTL restricted to H-2Kb. These data suggest that the predominance of isolation of H-2Db-restricted CTL clones from long term in vitro cultures may be a function of the relative affinity of this population as a whole, rather than due to the immunodominance of this subpopulation during the in vivo response to SV40 T antigen.

Growth-Related expression of a 72,000 molecular weight poly(A) + mRNA binding protein

In this communication, we have studied a 72,000 mol w (p72) host protein which reacts with a mouse monoclonal antibody (PAb6) directed against antigenic determinants on the Simian virus 40 (SV40) large T antigen protein that map 5′ of 0.42 map units on the viral genome. The p72 protein is an abundant basic (p I>7) cytoplasmic protein found in both SV40-transformed and untransformed parental cells and in cell lines derived from normal human and tumor tissue. By two-dimensional gel analysis and Western blot analysis the p72 protein identified by PAb6 is indistinguishable from the 72,000 mol w protein PABP associated with the poly(A) + tract of cytoplasmic messenger RNA molecules. In normal human peripheral blood mononuclear cells stimulated to proliferate with the T-cell-specific mitogenic lectin phytohemagglutinin the synthesis and cytoplasmic accumulation of p72 occurs very early during the G 0→G 1-phase transition. The p72 protein is also expressed in proliferating and differentiated human promyelocytic HL60 cells indicating that the expression of this protein is not strictly limited to cycling cells.

Transformation of human cells by a polyomavirus containing complementing JCV and RFV genomes

Molecularly cloned viral DNA from late RFV (L-RFV) and early JCV (E-JCV) were transfected into human fetal brain (HFB) cells and complementation was demonstrated. A new infectious virus (E-JCV/L-RFV) was produced. Infection resulted in partial transformation of HFB and human embryonic kidney cells. No transformation was observed with EL-JCV or wtJCV. The transformants contained T-antigen and had a lifespan similar to SV40-transformed human cells but failed to express some phenotypes of transformation. All transformants contained E-JCV viral DNA, usually both integrated and episomal. Although no L-RFV DNA was present in the transformants, L-RFV appears to play a role in the initiation of transformation.

Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells

The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.

Genotoxic activities of benzamidine and its N-hydroxylated metabolite benzamidoxime in Salmonella typhimurium and mammalian cells.

The genotoxic potentials of benzamidine and benzamidoxime were determined to study the toxicological relevance of the metabolic N-oxygenation (N-hydroxylation) of benzamidines to benzamidoximes. Benzamidoxime induced DNA single-strand breaks (in rat hepatocytes) and DNA amplification in SV40-transformed hamster cells. In the experiments performed, benzamidine itself was only marginally positive in the hepatocyte/DNA single-strand break assay. Since these cells possess an intact metabolization apparatus, the biological activities may be attributed to toxic and genotoxic metabolites formed by biotransformation. In the Salmonella typhimurium mutagenicity test (TA 98 and TA 100) benzamidoxime alone exhibited a low mutagenicity in the TA 98 strain in the presence of rabbit liver S-9 fractions. These results permit recognition of the metabolic N-hydroxylation of benzamidines to benzamidoximes as a process to toxication. Indirect evidence for the formation of a glucuronide of benzamidoxime has been obtained from in vitro experiments, but it could not be established that this process was a decisive factor in the genotoxicity of benzamidoxime.

Induction of simian virus 40 transplantation immunity in hamster by gel-purified SV40 large T antigen

Simian virus 40 (SV40) large T antigen and p53 cellular protein were isolated from an SV40-transformed hamster cell line by immunoprecipitation with anti-T sera and purified by sodium dodecyl sulfate—gel electrophoresis. These two protein were tested in hamsters for the presence of SV40 transplantation rejection antigenic sites by in vivo transplantation rejection assay. The large T antigen immunized the hamsters against a challenge of SV40 tumor cells and the protected animals generated cytotoxic spleen cells. Hamsters immunized with the p53 cellular protein were not protected against SV40-induced tumor but there was some delay in the appearance of tumor.

Target-induced changes in macrophage migration may explain differences in lytic sensitivity among simian virus 40-transformed fibroblasts.

Time-lapse video microscopy has been used to study macrophage movement in the presence of SV40-transformed fibroblasts. In all, five different SV40-transformed cell lines (an uncloned line and four clones derived from it) were tested for their affects on the movement of LPS-activated macrophages (AM). Conditions for video microscope recording were designed to simulate, as best as possible, the conditions used in a 51Cr-release cytotoxicity assay. Our analysis shows that these targets had a range of effects on macrophage migration, from stimulation to complete inhibition of movement. Two of the targets we tested (SV-COL and SV-COL-E8) both highly sensitive to lysis, stimulated macrophage movement, inducing an "agitated" response. Two other targets (SV-COL-F11 and SV-COL-H5), both of intermediate sensitivity had, for the most part, a suppressive effect on macrophage movement. Finally, we found that one target, clone SV-COL-F5, which is completely resistant to lysis by AM, was able to completely inhibit macrophage movement. This inhibitory effect was accompanied by a "pathologic" change in the structure of the macrophage cytoplasm suggesting that this target escapes lysis in vitro by secreting a factor that incapacitates the effector cell. Overall, these observations suggest that target cell products that can affect the behavior of the AM may be important in determining the lytic sensitivity of transformed target cells.

In vitro selection of SV40 T antigen epitope loss variants by site-specific cytotoxic T lymphocyte clones.

SV40-transformed cells of C57BL/6 (B6) mouse origin (H-2b) express four distinct predominant antigenic sites, I, II, III, and IV, on SV40 large tumor (T) Ag that are recognized by SV40 T Ag-specific CTL clones. In this study, we selected SV40 T Ag-positive cell lines which had lost one or more of the antigenic sites, by in vitro cocultivation of a SV40-transformed B6 mouse kidney cell line (K-0) with SV40 T Ag site-specific CTL clones, Y-1 (site I specific), Y-2 (site II specific), Y-3 (site III specific), and Y-4 (site IV specific). All of the CTL-resistant cell lines expressed large quantities of cell surface H-2 class I Ag. K-1 cells selected by CTL clone Y-1 lost the expression of antigenic sites I, II, and III, but not site IV. K-2 and K-3 cells selected by CTL clones Y-2 and Y-3, respectively, were found to be negative for sites II and III but expressed sites I and IV. K-4 cells selected by CTL clone Y-4 lost the expression of only site IV. K-1,4 cells (sites I-, II-, III-, IV-) were selected from K-1 cells by cocultivation with CTL clone Y-4, K-2,4 cells (sites I+, II-, III-, IV-) were selected from K-2 cells by CTL clone Y-4. K-3,1 cells (sites I-, II-, III-, IV+) were selected from K-3 cells by CTL clone Y-1, and K-3,1,4 cells (sites I-, II-, III-, IV-) were selected from K-3,1 cells by CTL clone Y-4. From K-4 cells, K-4,1 cells (sites I-, II-, III-, IV-) and K-4,3 cells (sites I+, II-, III-, IV-) were selected by CTL clone Y-1 and Y-3, respectively. The antigenic site loss variant cell lines K-1, K-1,4, K-3,1 K-3,1,4, K-4,1, and K-4,3 synthesized SV40 T Ag molecules of 75, 75, 78, 78, 81, and 88 kDa, respectively. Expression of wild-type SV40 T Ag in the antigenic site loss variants by infection with SV40 or transfection with cloned SV40 DNA restored the CTL recognition sites on the variant cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ataxia telangiectasia resists gene cloning: an account of parameters determining gene transfer into human recipient cells.

A subclone of an SV40-transformed fibroblast cell line from a patient with Ataxia telangiectasia (AT) with a relatively high rate of DNA uptake was isolated. However, more than 65000 independent genomic transfectants (using wild-type human DNA) did not contain the functional AT gene. This number represents the statistical distribution of an amount of DNA equivalent to more than three times the haploid human genome. The transfectants were screened by an X ray selection protocol that could rescue a single wild-type cell out of a population of 10(6) AT cells. This suggests a reversion frequency for AT of below 10(-8). The DNA uptake into human cells is compared with that into NIH3T3 cells and future possibilities for the isolation of human repair genes are discussed.

Adeno-associated viruses inhibit SV40 DNA amplification and replication of herpes simplex virus in SV40-transformed hamster cells

Coinfection with HSV-1 and any of the three defective parvoviruses AAV-2, AAV-3, or AAV-5 reduces the HSV-1-induced amplification of SV40 DNA in the SV40-transformed hamster cell lines C0631 and Elona. Moreover, all three viruses significantly decrease HSV-1 replication. The effects are measurable to the same extent in both cell lines and are dependent on the quantity of AAV DNA molecules rather than on infectious AAV particles. It seems unlikely that the inhibitions are due to simple competition but result from complex interactions that involve the terminal sequences of AAV DNA.

Establishment and characterization of rat pancreatic beta cell lines transformed by simian virus 40.

Rat pancreatic beta cells were transformed by simian virus 40 (SV40). The established beta cell lines expressed tumor antigen specific to SV40 and retained the ability to produce immunoreactive insulin (IRI) even at a high passage level. One of 12 transformed beta cell lines, SV-PB1205, was further investigated in vitro and in vivo. Firstly, effects of drugs on replication of the pancreatic beta cells were examined in in vitro experiments. Various drugs increased the uptake of 3H-thymidine into the cells. Those included D-glucose, tetragastrin and secretin. However, such effect was not observed for glucagon and growth hormone. Secondly, the SV40-transformed pancreatic beta cells were transplanted into diabetic rats. This produced such improvement of plasma glucose level at least for 2 weeks. The Significance of those experiments was discussed.

Evidence that immunological variants of p53 represent alternative protein conformation

In normal murine lymphocytes the cellular oncoprotein, p53, exists as two immunologically distinct species which are reciprocally expressed in quiescent (p53-Go) and mitogenically stimulated (p53-G÷) cells. More recently, we have identifed discrete forms of p53, immunologically similar to p53-Go and p53-G÷, in 3T3 cells transformed by simian virus 40 (SV40). In this report, we demonstrate that immunologically distinct p53 species can also be expressed in vitro, from a single murine p53 cDNA clone. The p53 variants expressed in vitro and in SV40-transformed 3T3 cells have been studied by immunoprecipitation and Western blotting. Immunoprecipitation data indicated that, in their native conformations, the p53 variants react with either the PAb421 or RA3.2C2 monoclonal antibodies, but not with both. When analyzed by Western blotting, however, the denatured proteins were found to react with both monoclonal antibodies. This suggests that the p53 protein is flexible and can fold in alternative conformations so as to expose or mask different epitopes. We propose, therefore, that immunologically distinct p53 species are generated, at least partly, by conformational changes in a single polypeptide species.

Characterization of macrophage recognition and killing of SV40-transformed tumor cells that are "resistant" or "susceptible" to contact-mediated killing.

In this report we used the macrophage-"resistant" and -"susceptible" cell lines, F5m and F5b, to determine why AKR or AKR-like virus expression makes the F5m cell line more resistant to in vitro macrophage killing than the F5b cell line. We found that resistance to macrophage killing may be transmitted by an infectious AKR or AKR-like murine leukemia virus and that resistance was concomitant with virus expression as measured by the presence of AKR virus-specific 70 kDa glycoprotein. We report that macrophage cytotoxicity of these cell lines is dependent upon the direct contact between tumor cells and macrophages. In contrast, macrophage-mediated cytostasis occurred via soluble macrophage products and no differential susceptibility of F5b or F5m to macrophage-mediated cytostasis was observed. Macrophage binding of F5b was also significantly better than that of F5m. These data suggest that only the events that depend upon the close contact of macrophages and tumor cells will be affected by the expression of AKR or AKR-like virus. Therefore, the differences in susceptibility of F5m and F5b to direct macrophage-mediated cytotoxicity are apparently because the macrophage binding of F5m is less efficient than the binding of F5b.

"Transformation" of human endothelial cells by SV40 virions.

Human endothelial cells derived from the umbilical vein were transformed with SV40 virions. A cell line subcultured for over 60 serial passages was characterized in comparison with its untransformed counterpart which was culturable for less than five passages. The SV40-transformed human endothelial cells, designated SV-HUVEC, were positive not only for tumor (T) antigen specific to the SV40-transformed cell, but also for two markers of endothelial cells, Factor VIII-related antigen and a receptor for Ulex europaeus agglutinin I. By transformation the growth potential of the human endothelial cells was increased and their serum requirement was decreased. The SV40-transformed endothelial cells were, however, unable to form colonies in soft agar or to form tumors in athymic nude mice, although a small nodule was produced at the site of inoculation. Subcultivation of these cells up to the 62nd passage eventually resulted in crisis and loss of further cell division. Thus, the human endothelial cells were transformed by SV40 while retaining certain normal functions but without showing tumorigenicity.

Recognition of simian virus 40 T antigen synthesized during viral lytic cycle in monkey kidney cells expressing mouse H2K b- and H2D b-transfected genes by SV40-specific cytotoxic T lymphocytes leads to the abrogation of virus lytic cycle

Simian virus 40 (SV40)-encoded tumor or T antigen localizes in the membranes in addition to the nucleus of SV40-infected permissive monkey cells and SV40-transformed nonpermissive cells. The surface T antigen in SV40-transformed mouse cells provides a target for the cytotoxic T lymphocytes (CTL) which recognize SV40 T antigen in association with murine K/D, class I H-2 antigens. In order to demonstrate that SV40 T antigen synthesized in SV40-infected permissive monkey kidney cells (TC-7) may also function as a target for CTL, cloned murine H2D b and H2K b genes were expressed in TC-7 cells by DNA transfection and TC-7 cell lines expressing high levels of either H2K b or HD b antigens were established after cell sorting. SV40-infected TC-7/H2K b and TC-7/H2D b cells became susceptible to lysis by SV40-specific H-2 b restricted CTL. The susceptibility of these transfected SV40-infected monkey cells to anti-SV40 bulk culture CTL and SV40-specific H2D b- and H2K b-restricted CTL clones depended upon the synthesis of SV40 T antigen and the expression of the appropriate H2K b or H2D b restriction elements. Treatment of SV40-infected TC-7/H2D b and TC-7/H2K b With CTL clones abrogated the virus lytic cycle indicating that CTL may play an important role in limiting papovavirus infection in the natural host.

Various short-term assays and two long-term studies with the plasticizer di(2-ethylhexyl)phthalate in the Syrian golden hamster.

The plasticizer di(2-ethylhexyl)phthalate (DEHP) and its main metabolite monethylhexylphthalate (MEHP) were investigated in several short-term in vitro assays, including mutagenicity in Salmonella typhimurium TA102, a strain sensitive to mutations arising as a cause of oxidative DNA damage. Also DNA amplification in SV40-transformed Chinese hamster cells and DNA damage in rat and hamster hepatocytes were investigated. The two compounds were not genotoxic in any of the test systems. Furthermore, DEHP was investigated in two long-term bioassays with Syrian golden hamsters using both i.p. (max. total dose 54 g/kg) and inhalative (7-10 mg/kg) application. In both experiments an additional group of animals received a combination treatment of DEHP with N-nitrosodimethylamine (NDMA). These studies were included in order to elucidate whether the observed influence of DEHP on the microsomal enzyme activity (Seth, 1982) may effect the carcinogenic activity of NDMA. There was no significant increase in tumor incidence after application of only DEHP via both routes. However, the occurrence of liver malignancies was significantly (P less than 0.001) reduced after the combination treatment in the inhalation study.

The expression of the Escherichia coli uvrA gene in human cells

Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously. The E. coli gpt gene in pSV2 gpt is used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. SV40-transformed XP-A cells were transfected with pSV2 uvrASV2 gpt, gpt + colonies were selected, and cell lines esttablished. Several times were examined in detail. Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95 000-dalton UvrA-related protein. The expression of uvrA was found to be 50–100-fold lower than the expression of gpt. Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities. The peptide maps derived from partial proteolysis of the “mammalian” UvrA protein are identical to the E. coli UvrA protein. The sub-cellular location of UvrA protein in uvrA + XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear. Two uvrA + cell lines were examined for their UV_resistant phenotye and not unexpectedly were found not to be reverted to a state of repair proficiency.

SV40-transformed human fibroblasts exhibit a characteristic chromosomal pattern.

A karyotype study of seven SV40-transformed human fibroblast cell lines was performed using R-banding. Although large variations existed from line to line and, to a lesser degree, from cell to cell in a given line, many common features were found. The most characteristic were chromosome imbalances. Some of the chromosomes or chromosome segments present in excess were, in decreasing order of frequency, the early replicating X, 12q, 3q, 12p, 19, 1p, and 6p. Losses of other chromosomes and chromosome segments were also frequent and involved 11p, 2p, 6q, 10p, 18, 4q, 8p, 4p, 10q, and 16. These imbalances seemed to correlate with metabolic characteristics, previously described, such as low activities of catalase (11p), superoxide dismutase 2 (6q), and acid phosphatase (2p, 11p) and a high ratio of lactate dehydrogenase B (LDHB, 12p) activity to LDHA (11p) activity.

Modulation of p53 protein expression during cellular transformation with simian virus 40.

We analyzed the relation of metabolic stabilization of the p53 protein during cellular transformation by simian virus 40 (SV40) to (i) expression of the transformed phenotype and (ii) expression of the large tumor antigen (large T). Analysis of SV40-tsA28-mutant-transformed rat cells (tsA28.3 cells) showed that both p53 complexed to large T and free p53 (W. Deppert and M. Haug, Mol. Cell. Biol. 6:2233-2240, 1986) were metabolically stable when the cells were cultured at 32 degrees C and expressed large T and the transformed phenotype. At the nonpermissive temperature (39 degrees C), large-T expression is shut off in these cells and they revert to the normal phenotype. In such cells, p53 was metabolically unstable, like p53 in untransformed cells. To determine whether metabolic stabilization of p53 is directly controlled by large T, we next analyzed the metabolic stability of complexed and free p53 in SV40 abortively infected normal BALB/c mouse 3T3 cells. We found that neither p53 in complex with large T nor free p53 was metabolically stable. However, both forms of p53 were stabilized in SV40-transformed cells which had been developed in parallel from SV40 abortively infected cultures. Our results indicate that neither formation of a complex of p53 with large T nor large-T expression as such is sufficient for a significant metabolic stabilization of p53. Therefore, we suggest that metabolic stabilization of p53 during cellular transformation with SV40 is mediated by a cellular process and probably is the consequence of the large-T-induced transformed phenotype.