Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.     

Surface T-antigen expression in simian virus 40-transformed mouse cells: correlation with cell growth rate.

Cell growth control appears to be drastically altered as a consequence of transformation. Because the cell surface appears to have a role in modulating cell growth and simian virus 40 (SV40)-transformed cells express large T antigen (T-Ag) in the plasma membrane, we investigated whether surface T-Ag expression varies according to cell growth rate. Different growth states were obtained by various combinations of seeding density, serum concentration, and temperature, and cell cycle distributions were determined by flow microcytofluorometry. Actively dividing SV40-transformed mouse cell cultures were consistently found to express higher levels of surface T-Ag and T-Ag/p53 complex than cultures in which cells were mostly resting. In addition, the T-Ag/p53 complex disappeared from the surface of tsA7-transformed cells cultured under restrictive conditions known to induce complete growth arrest (39.5 degrees C), although the surface complex did not disappear from other tsA transformants able to keep cycling at 39.5 degrees C. These results suggest that surface SV40 T-Ag or surface T-Ag/p53 complex, or both, are involved in determining the growth characteristics of SV40-transformed cells.     

"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.     

SV40 T基因转化的山羊乳腺上皮细胞系及其生物学特性

目的建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系.方法根据已发表的SV40病毒T基因序列设计引物,以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板,用高保真PCR扩增SV40 T基因.将获得的SV40 T基因克隆入真核表达载体,并用获得的重组表达质粒转染山羊原代乳腺上皮细胞.经有限稀释和反复传代后获得转化细胞克隆,对其生物学特性进行研究.结果扩增出序列正确的SV40T基因,重组质粒转染获得的转化细胞的对数生长期为接种后第4天,细胞群体倍增时间为23.5*!h,克隆形成率为26.7%.DNA斑点杂交试验证明转化细胞的基因组中整合有SV40 T基因,染色体核型分析试验表明转化细胞的核型无明显异常,裸鼠接种试验证明转化细胞不能形成肿瘤,软琼脂集落形成试验表明转化细胞在软琼脂中不能生长.部分细胞克隆已在体外传30代以上,保持正常乳腺上皮细胞的形态特征,在胶原基质上能形成腺泡样结构.结论本研究获得的SV40 T基因转化的山羊乳腺上皮细胞具有转化细胞系的生物学特性.     

SV40 large T-antigen and transformation related protein p53 are associated in situ with nuclear RNP structures containing hnRNA of transformed cells

The localization of SV40 large T-antigen (T-Ag) and the cellular protein p53 in the nuclei of mouse and human SV40-transformed cells and of a methylcholanthrene-transformed mouse cell line, was studied. Their detection by ultrastructural immunocytochemistry with specific monoclonal antibodies employed two complementary methods used in parallel. These consisted of indirect immunoperoxidase labelling carried out before embedment on Triton-permeabilized cells, or indirect immunogold labelling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that in SV40-transformed cells both proteins are chiefly localized on peri- and interchromatin RNP fibrils. This shows that they occur in structures involved in the synthesis and processing of hnRNA. The nucleoli and chromatin did not appear to be labelled. In methylcholanthrene-transformed cells the protein p53 (in the absence of large T-Ag) was also detected on peri- and interchomatin fibrils. Taken together with recent results which demonstrated that, during lytic infection, T-Ag was associated chiefly with cellular chromatin (Harper, F, Florentin, Y & Puvion, E, Exp cell res 161 (1985) 434) [33], our experiments provide evidence that the transforming function of SV40 large T-Ag is dissociable from its function in SV40 lytic infection in terms of its subnuclear distribution.     

Tumorigenicity of revertants from an SV40-transformed line

A syndrome of in vitro properties correlates with the tumorigenicity of SV40-transformed rodent cells. These properties are plasminogen activator production, loss of large actin cables, and anchorage-independent growth. An established rat fibroblast line, its SV40 transformant, several T-antigen negative revertants, and a spontaneous retransformant isolated form one of the revertants were analyzed in vivo for their tumorigenicity and in vitro for the syndrome. The two transformed lines were highly tumorigenic, and had clearly abnormal in vitro properties. The parental rat line was weakly tumorigenic in nude mice and demonstrated a slightly transformed response in the in vitro assays. The revertants were completely nontumorigenic. Expression of the in vitro syndrome was not uniform for all revertants; however, most cell lines maintained the correlation of the syndrome and tumorigenicity.     

Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.

SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.     

Reactivity to SV40 T antigen in athymic (nude), anti-thymocyte serum-treated, and normal mice.

Antisera prepared in syngeneic mice by hyperimmunization with intact SV40-transformed mouse cells or with somatic cell hybrids between SV40-transformed human and normal mouse cells exhibit anti-SV40 tumor (T) antigen reactivity. Athymic mice bearing tumors formed by SV40-transformed mouse, human or mouse-human hybrids were not reactive with SV40 T antigen. Anti-thymocyte serum (ATS)-treated mice also lacked T antigen reactivity during suppressive treatment but developed antibody to T antigen after discontinuing ATS treatment and tumor regression. We conclude that that presence of growing tumors in the mouse is not necessary for the production of anti-SV40 T antigen antibodies but that helper thymus-derived cells are essential for the humoral response.     

Idiotype network components are involved in the murine immune response to simian virus 40 large tumor antigen

Summary Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag), a monoclonal antibody specific for SV40 T-Ag (Ab-1 preparation), and a monoclonal anti-idiotypic antibody (anti-Id), designated 58D, were used to analyze the humoral immune response of Balb/c mice either immunized with recombinant SV40 T-Ag or challenged with SV40-transformed cells. Inhibition assays indicated that antibodies from mice immunized with SV40 T-Ag and from those bearing SV40 tumor inhibited the SV40 T-Ag/Ab-1 reaction. These data suggested that the antibody response in immunized or tumorchallenged mice recognized similar epitope(s) on SV40 T-Ag to that detected by the monoclonal Ab-1. These anti-(SV40 T-Ag) response antibodies also inhibited the Ab-1/anti-Id reaction and recognized the anti-Id in direct binding assays. Together, these data indicate that murine anti-(SV40 T-Ag) responses shared an idiotope with a monoclonal anti-(SV40 T-Ag) Ab-1 preparation. This idiotope, which is recognized by the monoclonal anti-Id preparation, 58D, appears to be involved in the humoral immune response to SV40 T-Ag in both SV40-T-Ag-immunized and tumor-bearing mice. The monoclonal anti-Id preparation may represent a focal point for manipulating the humoral immune response to tumors induced by SV40-transformed cells.     

Antibody response to simian virus 40 tumor antigen in nude mice reconstituted with T cells.

Athymic BALB/c nude mice (nu/nu) fail to generate circulating antibodies to simian virus 40 (SV40) tumor (T) antigen when immunized with SV40-transformed mouse cells or with T antigen positive somatic cell hybrids derived from SV40-transformed human and normal mouse parental cells. However, normal BALB/c mice readily produce antibodies to SV40 T antigen. When nude mice were reconstituted with normal syngeneic T lymphocytes from spleen or thymus source, the humoral immune responsiveness to SV40 T antigen was restored.     

Generation of cytotoxic lymphocytes by SV40-induced antigens

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

Genetic analysis of tumorigenesis. VIII. Suppression of SV40 transformation in cell hybrids and cytoplasmic transferants

Intraspecies somatic cell hybrids of BALB/c mouse 3T3 and SV40-transformed embryonic fibroblast (SVT2) cells were analyzed for transformation-associated properties and their tumorigenic potential in nude mice. In confirmation of our earlier findings, hybrids expressing the viral T-antigen were not suppressed for the ability to clone in medium with 1% serum. In contrast, division rate in medium with 1% or 10% serum, anchorage independence, cytochalasin-sensitive growth control, and tumorigenicity were suppressed noncoordinately, and the extent of suppression varied from one hybrid to another. Suppression was not simply determined by the increased chromosome content of the hybrid cells, nor was suppression correlated with rearrangements of the integrated viral sequence (SAGER et al., 1981a, b). Similar results were found in cytoplasmic transferants expressing T-antigen. Four independent transferants and subclones derived from them varied in the extent of suppression of anchorage independence and tumorigenicity. In both hybrids and transferants, a low serum requirement for clonal growth apparently was determined solely by expression of SV40 T-antigen, but other transformation properties, as well as tumorigenicity, appeared to require multiple changes in the cellular genome for their expression. These changes must occur during or after viral integration, since they are not expressed in uninfected 3T3 cells.     

Activation of endogenous retroviral transcription in SV40-transformed mouse cells.

We have previously isolated a number of cDNA clones that correspond to mRNAs present at higher levels in SV40-transformed cells than in the untransformed parental cells (Scott, M.R.D., Westphal, K.-H. and Rigby, P.W.J. (1983) Cell 34, 557-567). We have now determined the nucleotide sequence of the archetypal Set 2 clone, pAG59, and can thus identify it as corresponding to the env gene of the endogenous, ecotropic C-type retrovirus of Balb/c mice, Emv-1. We have shown that in the subset of SV40-transformed cells that express the provirus both of the proteins encoded by env, gp70 and p15E, are synthesised and that the former is displayed on the cell surface. We discuss the significance of these observations for the biology of SV40 transformation.     

Susceptibility of primate-mouse hybrid cells to SV40

Primate-mouse hybrid cells were challenged with SV40 DNA and monitored for their ability to produce virus. All of the hybrid cells had lost at least half of their primate chromosomes at the time of challenge. Only SV40 T-antigen-positive hybrid cells derived from an SV40-transformed human parental cell produced SV40. This finding suggests that the chromosome(s) necessary for SV40 replication are easily lost on fusion of mouse and primate cells unless the parental cells are already SV40-transformed.     

Impaired CK1 delta activity attenuates SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo

Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data show that inactivating mutations in CK1δ impair SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo.     

Antigenic and Immunogenic Characteristics of Nuclear and Membrane-Associated Simian Virus 40 Tumor Antigen

Antisera were prepared in syngeneic hosts against subcellular fractions of simian virus 40 (SV40)-transformed cells (MoalphaPM, MoalphaNuc), glutaraldehydefixed SV40-transformed cells (HaalphaH-50-G, MoalphaVLM-G), and electrophoretically purified denatured SV40 tumor antigen (T-ag) (RaalphaT). Immune sera were also collected from animals bearing tumors induced by SV40-transformed cells (HaalphaT, MoalphaT, HAF) and from SV40-immunized animals that had rejected a transplant of SV40-transformed cells (HaalphaS, MoalphaS). Immunological reagents prepared against cell surface (MoalphaPM, HaalphaS, MoalphaS, HaalphaH-50-G, MoalphaVLM-G) reacted exclusively with the surface of SV40-transformed cells by indirect immunofluorescence or protein A surface antigen radioimmunoassay. Immunological reagents prepared against the nuclear fraction (MoalphaNuc) or whole-cell determinants (HaalphaT, MoalphaT, HAF, RaalphaT) reacted with both the nuclei and surface of SV40-transformed or -infected cells. All reagents were capable of immunoprecipitating 96,000-molecular weight large T-ag from solubilized whole cell extracts of SV40-transformed cells. The exclusive surface reactivity of HaalphaS exhibited in immunofluorescence tests was abolished by solubilization of subcellular fractions, which then allowed immunoprecipitation of T-ag by HaalphaS from both nuclear and plasma membrane preparations. Specificity was established by the fact that all T-reactive reagents failed to react in serological tests against chemically transformed mouse cells, and sera from mice bearing transplants chemically transformed mouse cells (MoalphaDMBA-2) failed to react with SV40-transformed mouse or hamster cells. Reagents demonstrating positive surface immunofluorescence and protein A radioimmunoassay reactions against SV40-transformed cells were capable of blocking the surface binding of RaalphaT to SV40-transformed cells in a double-antibody surface antigen radioimmunoassay. This blocking ability demonstrated directly that a component specificity of each surface-reactive reagent is directed against SV40 T-ag. A model is presented which postulates that the differential detection of T-ag by the various serological reagents is a reflection of immunogenic and antigenic differences between T-ag polypeptides localized in nuclei and plasma membranes.     

Noncoordinate expression of SV40-induced transformation and tumorigenicity in mouse cell hybrids.

Somatic mouse cells hybrids formed by fusion of nontumorigenic 3T3 closely related SV40-transformed SVT2 cells were analyzed in a study designed to probe the genetic basis of the multiple phenotypic changes induced by SV40 transformation. These hybrids showed noncoordinate expression of the transformation phenotype. Although they cloned at high efficiency in medium with low serum and expressed the SV40 T-antigen of the SVT2 parent, hybrid cells grew poorly without anchorage and exhibited a cell and colony morphology intermediate between that of the parents. Tumorigenicity was assayed quantitatively by subcutaneous coinjection into athymic nude mice of serial dilutions of 10(2) to 10(5) hybrid cells with 10(7) lethally irradiated 3T3 cells. The results showed that 100--1000 times more hybrid cells had to be injected for tumor formation than were required with SVT2. These and other observations show that most 3T3/SVT2 hybrid cells are not tumorigenic but that each population contains a rare subset of tumorigenic cells.     

Evidence for the involvement of cytolytic macrophages in rejection of SV40-induced tumors

The potential role of cytolytic macrophages in in vivo resistance to tumors induced by simian virus 40 (SV40) was evaluated in two experimental systems. First, a cell line produced by sequential in vivo passage of SV40-transformed fibroblasts through syngeneic C3H/HeJ mice was found to develop both increased neoplastic character and resistance to macrophage-mediated lysis, suggesting in vivo selection pressure against the macrophage-sensitive phenotype. In the second approach, SV40-transformed cells from C3H.OL mice, a strain that fails to produce SV40-specific cytolytic T lymphocytes (CTL), were cloned, and the cloned cells were tested for susceptibility to macrophage cytolysis in vitro. Two clones SV-COL-E8 and SV-COL-F5, which represent the extremes of macrophage susceptibility and resistance, respectively, were tested for progressive growth in syngeneic C3H.OL recipients. Progression in vivo was found to correlate with resistance to macrophage cytolysis in vitro. Other in vitro measures of the neoplastic phenotype, cell division rate and anchorage-independent growth, did not predict the relative abilities of clones E8 and F5 to form tumors. Likewise, the cells were indistinguishable in their sensitivity to cytolysis by allogeneic CTL and by natural killer cells. Finally, the presence of activated macrophages in the peritoneum of mice rejecting a challenge of syngeneic SV40-transformed cells was confirmed in both CTL responder and nonresponder strains. These studies suggest that cytolytic macrophages are indeed generated during rejection of SV40-induced mouse tumors and that, in the absence of an effective anti-SV40 CTL response, resistance of the transformed cell to macrophage-mediated cytolysis can be a determining factor in in vivo tumor growth.     

Comparison of simian virus 40 large T antigen recombinant protein and DNA immunization in the induction of protective immunity from experimental pulmonary metastasis

In this report we examine the ability of a recombinant tumor antigen preparation to prevent the establishment of experimental pulmonary metastasis. Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was injected into BALB/c mice followed by challenge with an intravenous injection of syngeneic SV40-transformed tumorigenic cells. The experimental murine pulmonary metastasis model allows for the accurate measurement of metastatic lessions in the lungs at various times after the challenge, using computer-assisted video image analysis. Following challenge, lung metastasis and survival data for the groups of mice were obtained. Animals immunized with recombinant SV40 T-Ag showed no detectable sign of lung metastasis and survived for more than 120 days after challenge. Antibodies specific for SV40 T-Ag were detected in the serum of immunized mice by enzyme-linked immunosorbent assay. Splenocytes obtained from mice immunized with recombinant SV40 T-Ag did not lyse syngeneic tumor cells, indicating that no cytotoxic T lymphocyte response was induced. Control mice developed extensive lung metastasis and succumbed to lethal tumor within 4 weeks after challenge. These data indicate that immunization with the recombinant SV40␣T-Ag induces protective, T-Ag-specific immunity in an experimental pulmonary tumor metastasis model. Received: 20 August 1998 / Accepted: 25 November 1998     

Expression of malignancy traits in the interspecific somatic hybrids of tumor and normal cells

Tumorigenicity and anchorage independence in two types of the interspecies hybrids of the tumor and normal mammalian cells were studied. One hybrid type was derived from fusion of spontaneously transformed Chinese hamster and normal mouse cells; the second type was obtained by fusion of SV40-transformed Djungarian hamster and the same mouse cells. The tumorigenicity in the athymic nude mice was suppressed in the first type of hybrids. The hybrid clones derived from fusion of SV40-transformed and normal cells could form tumor in nude mice. Testing of hybrid clones for their ability to form colonies in soft agar showed that all hybrids grew well in the medium, similar to tumor parental cells. These data suggest that malignancy and anchorage independence are under separate genetic control. The influence of the origin of the tumor parental cells (spontaneous or SV40-virus transformation) on the expression of the malignancy in hybrids of the tumor and normal cells is discussed.