SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.     

Inhibition of simian virus 40 T-antigen expression by cellular differentiation.

Murine 3T3T stem cells transfected with pSV3neo DNA were employed to study the effects of somatic cell differentiation on simian virus 40 (SV40) T-antigen expression. This experimental approach was used because the 3T3T cell line is a well-characterized in vitro adipocyte differentiation system and the pSV3neo plasmid contains the early region of the SV40 genome and a selective marker, G418 resistance. Cell clones containing stably integrated pSV3neo which expressed T antigen were isolated in G418-containing medium. Most of these cell clones differentiated poorly. However, several clones retained the ability to efficiently differentiate into adipocytes, and with these cell clones, it was established that adipocyte differentiation markedly repressed T-antigen expression. The differentiation-specific repression of T-antigen expression did not result from a loss of proliferative potential associated with terminal differentiation, because it was observed in adipocytes that could be restimulated to proliferate. In such cells, restimulation of cell growth induced reactivation of T-antigen expression. Repression of T-antigen expression was also demonstrated during differentiation of SV40 T-antigen-immortalized human keratinocytes. These results establish that the process of cellular differentiation can repress T-antigen expression in at least two distinct biological systems.     

Influence of transfected SV40 early region on growth and differentiation of human endothelial cells.

Endothelial cells isolated from human umbilical veins show a limited in vitro life span of about 40 population doublings. Cell division is dependent on the presence of endothelial cell growth factor in the culture medium. We have transfected primary endothelial cells with a plasmid containing the early region of SV40 virus. Large T positive cells were obtained which grew in the absence of endothelial cell growth factor at low serum concentrations and showed a prolonged lifespan. Expression of von Willebrand factor and SV40 large T antigen was detected simultaneously in transfected cells.     

Immortalization of human fibroblasts using tsA mutant of SV40 and pSV3neo plasmid]

Clones of immortalized human fibroblasts with an extended life span in culture and a capability of subloning were obtained after the infection with a temperature sensitive mutant (tsA 239) of SV40 virus and pSV3neo plasmid. As compared with the parental cells, the obtained clones exhibited increased plating efficiency, decreased doubling time, and serum dependence. We did not obtained the colony formation during cultivation of immortalized cells in semiliquid agar. This means that our cells were not completely malignant. The PCR (polymerase chain reaction)-analysis has revealed the presence of viral DNA at early passages (25th passage) after the infection by tsA SV40, and its absence after a prolonged cultivation (46th passage). PCR-analysis of the clones obtained after pSV3neo transfection has revealed the presence of gene A sequences either at early (9-15), or later (62) passages. The expression of the gene A product in cells of these clones was revealed only early passages (11 and 35). Possible mechanisms of immortal phenotype origin in human diploid cells after the action of ts-mutant and other constructions of SV40 are discussed.     

The genomic instability associated with integrated simian virus 40 DNA is dependent on the origin of replication and early control region.

DNA rearrangements in the form of deletions and duplications are found within and near integrated simian virus 40 (SV40) DNA in nonpermissive cell lines. We have found that rearrangements also occur frequently with integrated pSV2neo plasmid DNA. pSV2neo contains the entire SV40 control region, including the origin of replication, both promoters, and the enhancer sequences. Linearized plasmid DNA was electroporated into X1, an SV40-transformed mouse cell line that expresses SV40 large T antigen (T Ag) and shows very frequent rearrangements at the SV40 locus, and into LMtk-, a spontaneously transformed mouse cell line that contains no SV40 DNA. Stability was analyzed by subcloning G-418-resistant clones and examining specific DNA fragments for alterations in size. Five independent X1 clones containing pSV2neo DNA were unstable at both the neo locus and the T Ag locus. By contrast, four X1 clones containing mutants of pSV2neo with small deletions in the SV40 core origin and three X1 clones containing a different neo plasmid lacking SV40 sequences were stable at the neo locus, although they were still unstable at the T Ag locus. Surprisingly, five independent LMtk- clones containing pSV2neo DNA were unstable at the neo locus. LMtk- clones containing origin deletion mutants were more stable but were not as stable as the X1 clones containing the same plasmid DNA. We conclude that the SV40 origin of replication and early control region are sufficient viral components for the genomic instability at sites of SV40 integration and that SV40 T Ag is not required.     

Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.     

Extended life span of human endometrial stromal cells transfected with cloned origin-defective, temperature-sensitive simian virus 40.

Human endometrial stromal cells transfected with an origin-defective, temperature-sensitive simian virus 40 recombinant plasmid are dependent on T-antigen function for proliferation and at the permissive temperature have an extended life span in culture. Southern blot analysis indicates that the transfected gene is present in low copy number, possibly at a single integration site. Normal stromal cells are capable of 10 to 20 population doublings in culture. Transfected cultures have been carried at the permissive temperature to 80 population doublings before crisis. In the multistep model of malignant transformation of human cells, these cells represent one of the earliest stages: extended but finite life span. We have used these cells to investigate alterations in signal transduction that may be responsible for this early stage of transformation caused by the large T antigen. Temperature shift experiments indicate that the expression of ornithine decarboxylase (ODC) but not of c-fos is altered by the large T antigen. Induction of c-fos by serum or 12-O-tetradecanoylphorbol-13-acetate is independent of temperature. However, in the transfected cells, the induction of ODC by asparagine or serum is greatly enhanced at the permissive temperature. This result indicates that the large T antigen acts downstream of c-fos but upstream of ODC expression in the signal-transducing cascade.     

东方田鼠胚胎成纤维细胞永生化细胞系的建立

目的建立东方田鼠胚胎成纤维永生化细胞系,为全面研究东方田鼠抗日本血吸虫机制以及开展不同动物成纤维细胞间比较研究奠定基础和提供细胞实验材料。方法运用脂质体介导的基因转染法将pSV3neo质粒导入第3代东方田鼠胚胎成纤维细胞,经G418筛选抗性克隆并扩大培养,建立永生化细胞系;用PCR检测细胞株中SV40T基因的整合,RT-PCR鉴定SV40T基因在转染细胞中的表达;绘制东方田鼠胚胎成纤维永生化细胞生长曲线。结果阳性细胞克隆已扩大培养并稳定传代50代,经鉴定SV40T抗原已整合到东方田鼠胚胎成纤维细胞中且稳定表达。结论成功建立东方田鼠胚胎成纤维永生化细胞系。     

Differentiation between senescence (M1) and crisis (M2) in human fibroblast cultures

Normal human fibroblasts undergo only a limited number of divisions in culture and eventually enter a nonreplicative state designated senescence or mortality stage 1 (M1). Expression of certain viral oncogenes, such as the SV40 large T antigen (SV40 T-Ag), can elicit a significant extension of replicative life span, but these cultures eventually also cease dividing. This proliferative decline has been designated crisis or mortality stage 2 (M2). BrdU incorporation assays are commonly used to distinguish between senescence (<5% labeling index) and crisis (>30% labeling index). It has not been possible, however, to ascertain whether the high labeling index, indicative of ongoing DNA replication, was caused by the presence of T-Ag. We used gene targeting to knock out both copies of the p21(CIP1/WAF1) gene in presenescent human fibroblasts. p21 -/- cells displayed an extended life span but eventually entered a nonproliferative state. In their terminally nonproliferative state both p21 +/+ and p21 -/- cultures were positive for the senescence-associated beta-galactosidase (SA-beta-gal) activity; in contrast, the labeling index of p21 +/+ cells was low (<5%) whereas the labeling index of p21 -/- cells was high (>30%). The observation that p21 -/- and SV40 T-Ag-expressing cells behave identically with respect to life span extension as well as the high labeling index in the terminally nonproliferative state indicates that crisis is not a phenomenon induced solely by viral oncogenes, but a physiological state resulting from the bypass of normal senescence mechanisms. The widely used biomarker for senescence, SA-beta-gal, cannot distinguish between senescence and crisis. We propose that all SA-beta-gal-positive cultures should be further examined for their BrdU labeling index.     

Human fibroblast commitment to a senescence-like state in response to histone deacetylase inhibitors is cell cycle dependent.

Human diploid fibroblasts (HDF) complete a limited number of cell divisions before entering a growth arrest state that is termed replicative senescence. Two histone deacetylase inhibitors, sodium butyrate and trichostatin A, dramatically reduce the HDF proliferative life span in a manner that is dependent on one or more cell doublings in the presence of these agents. Cells arrested and subsequently released from histone deacetylase inhibitors display markers of senescence and exhibit a persistent G1 block but remain competent to initiate a round of DNA synthesis in response to simian virus 40 T antigen. Average telomere length in prematurely arrested cells is greater than in senescent cells, reflecting a lower number of population doublings completed by the former. Taken together, these results support the view that one component of HDF senescence mimics a cell cycle-dependent drift in differentiation state and that propagation of HDF in histone deacetylase inhibitors accentuates this component.     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

Cellular senescence involves stochastic processes causing loss of expression of differentiated function genes: transfection with SV40 as a means for dissociating effects of senescence on growth and on differentiated function gene expression

In the accompanying work we demonstrated that the decline in expression of steroid 17 alpha-hydroxylase in mass cultures and clones of adrenocortical cells is the result of a stochastic switching process which yields mixtures of expressing and nonexpressing cells. There is an apparent positive correlation between the replicative potential of adrenocortical cell cultures and the number of cells in the culture that can express 17 alpha-hydroxylase. We investigated this by extending the cells' replicative potential by transfecting them with cloned SV40 virus. Cells from a senescent subclone, with very limited remaining replicative potential, were transfected. The cell population showed a progressive increase in growth rate and gave rise to a line of cells that expressed T antigen and which was apparently immortalized. Induction of mRNA for 17 alpha-hydroxylase by cyclic AMP was absent in this line of cells, as it was in the senescent cells prior to transfection. The cells remained responsive to gene induction by cyclic AMP as evidenced by increases in mRNA and activity for cholesterol side-chain cleavage. The absence of 17 alpha-hydroxylase expression in this line was not the result of interference by SV40 T antigen. When early passage cells were transfected with pSV3neo, which contains the early region of SV40 and neo, and were selected with G418, SV40 T antigen-expressing lines were derived which showed high levels of expression of 17 alpha-hydroxylase after induction with cyclic AMP. These cells maintained high levels of expression of 17 alpha-hydroxylase through four successive recloning events, over a period of replication much longer than that achievable by nontransfected cells. Thus, transfection by SV40 can be used to dissociate effects of senescence on growth and differentiated gene expression. T antigen expression selectively affects growth, but preserves the state of expression of a differentiated function gene as it was prior to transfection.     

Failure of simian virus 40 small t antigen to disorganize actin cables in nonpermissive cell lines.

Mouse C3H 10T1/2 cell lines expressing the simian virus 40 (SV40) small t antigen were obtained by cotransfection of pSV2neo and plasmids which encode small t. Cell lines derived from two plasmids which encode small t in the absence of stable deletion fragments of the large T antigen were morphologically normal and grew to slightly higher saturation densities in low serum than control cell lines. Unexpectedly, the clones had highly organized actin cables, as did parental 10T1/2 cells infected with wild-type SV40. These observations and comparisons of rat F111 cells infected with either polyomavirus or SV40 suggest that the SV40 small t antigen does not directly affect cytoskeletal organization.     

Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the “young” morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21^sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.     

A recombinant murine retrovirus for simian virus 40 large T cDNA transforms mouse fibroblasts to anchorage-independent growth.

A recombinant murine retrovirus containing the intact cDNA sequence for the simian virus 40 (SV40) large T antigen (T) was constructed by using the pZIPNeo SV(X)1 vector. Psi 2 packaging cells were then transfected, and G418-resistant clones were used to generate helper-free viral stocks. NIH 3T3 mouse fibroblasts infected by the recombinant T cDNA retrovirus were selected fro G418 resistance. Such cultures synthesized authentic SV40 T and were transformed to anchorage-independent growth at high efficiency. Therefore, this vector has allowed the study of the transformation properties of T under conditions of neutral drug selection and in the absence of SV40 small t antigen.     

Secretion of apolipoprotein A-I in lipoprotein particles following transfection of the human apolipoprotein A-I gene into 3T3 cells

Apolipoprotein A-I (apoA-I) is the major protein constituent of plasma high density lipoproteins (HDL). To examine apoA-I processing and secretion, the human apoA-I gene (2.2-kilobase PstI-PstI fragment) linked to the mouse metallothionein promoter was transfected by electroporation into NIH 3T3 fibroblasts along with the plasmid pSV2 neo, which confers neomycin resistance. Transfected cells were selected for neomycin resistance and screened for the ability to produce apoA-I by enzyme-linked immunosorbent assay. In the absence of lipids in the medium, selected 3T3 cells secreted apoA-I, mainly in the proprotein form, at density greater than 1.25 g/ml. Following incubation of cells with lipids, and subsequent washing with lipid-free medium, apoA-I was recovered in the HDL region (1.063-1.21 g/ml) as well as in the 1.21 g/ml infranatant. Examination of the HDL fraction by electron microscopy revealed round particles, 10-21 nm in diameter. These data indicate that human apoA-I secreted by transfected 3T3 fibroblasts can assemble into lipoprotein particles under the appropriate conditions.     

Study of the Functional Activities Concomitantly Retained by the 115,000 Mr Super T Antigen, an Evolutionary Variant of Simian Virus 40 Large T Antigen Expressed in Transformed Rat Cells

Simian virus 40 (SV40) transformed V 11 F 1 clone 1 subclone 7 rat cells (subclone 7) do not synthesize normal-size large T antigen (M(r), 90,000); instead, they produce a 115,000 M(r) super T antigen (115K super T antigen). This super T antigen is SV40 virus coded, and its synthesis results from rearrangement and amplification of integrated viral DNA sequences in subclone 7 (May et al., Nucleic Acids Res. 9:4111-4128, 1981). In this study the functional activities of 115K super T antigen were compared with the functional activities of SV40 large T antigen. Transfection experiments were performed with (i) cosmid SVE 5 Kb and plasmid pSVsT, both containing the super T antigen gene and (ii) plasmids pSV1 and pSV40, both containing the large T antigen gene. Transfection of pSVsT DNA or SVE 5 Kb DNA into secondary cultures of rat kidney cells induced the formation of transformed cell foci with an efficiency that was about 50% of the efficiency of pSV1 DNA or pSV40 DNA. Concomitant with the transforming activity, two other activities were also retained by super T antigen, namely, the ability to enhance the level of host cellular protein p53 and the capacity to bind to p53. In contrast, pSVsT and SVE 5 Kb DNAs were markedly deficient in the capacity to support tsA58 DNA replication in CV1-P cells at a nonpermissive temperature (41 degrees C), as shown by cotransfection experiments. The yield of virus produced in these experiments was 400-fold less than the yield obtained in parallel experiments with pSV40 or pSV1. However, SVE 5 Kb and pSVsT have a functional SV40 replication origin, as shown by their efficient replication in COS 1 cells which provided functional large T antigen. Super T antigen also possesses a specific affinity for sequences of SV40 viral origin. Our results suggest that under certain conditions, evolutionary changes in T antigen take place and that these changes could be restricted to the phenotypic requirement of maintaining a structure that is able to induce cell transformation, to form a complex with p53, and to enhance the cellular level of p53. Therefore, there appears to be a close relationship among the activities of T antigen involved in transforming cells, in binding to p53, and in enhancing the p53 cellular level. Moreover, this set of activities appears to be separable from the replicative ability of T antigen, based on the observation that 115K super T antigen is markedly defective for initiating viral DNA synthesis.     

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells.

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.     

Establishment of a functional ovine fetoplacental artery endothelial cell line with a prolonged life span

To study mechanisms governing fetoplacental vascular function, we have established a primary ovine fetoplacental artery endothelial (OFPAE) cell line. These OFPAE cells produce nitric oxide (NO), proliferate, and migrate in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF). To overcome the senescence crisis that this primary OFPAE cell line will eventually enter, we attempted to establish a functional OFPAE cell line with a prolonged life span by transfecting cells with plasmids containing a neomycin resistance gene and a simian virus 40 gene (SV40) expressing large T (T) and small t (t) antigens. The OFPAE cells at passage 8 were transfected. After neomycin selection, the surviving OFPAE (designated SV40 OFPAE) cells were expanded up to passage 80. Up to passage 30, these SV40 OFPAE cells maintained a morphology similar to untransfected OFPAE cells. Expression of T and t antigens in SV40 OFPAE cells was confirmed by immunocytochemistry. These SV40 OFPAE cells exhibited positive uptake of acetylated low-density lipoprotein (Ac-LDL) and positive staining for NO synthase 3 (NOS3) and formed capillary-like tube structures on Matrigel. Up to passages 20-23, these SV40 OFPAE cells proliferated (P < 0.05) and produced (P < 0.05) NO in response to both FGF2 and VEGF. Moreover, this cell proliferation stimulated by FGF2 and VEGF was dose-dependently inhibited (P < 0.05) by PD98059 (a selective mitogen-activated protein kinase 1 and 2 [MAP2K1/2, also termed MEK1/2] inhibitor) or by LY294002 (a selective phosphoinositide 3-kinase [PI3K] inhibitor). These data indicate that SV40 OFPAE cells, at least at passage 23, retain endothelial phenotypes and functions similar to their parental, untransfected OFPAE cells. Thus, a functional OFPAE cell line with an extended life span has been successfully established, potentially providing a valuable cell model for studying fetoplacental endothelial function.     

Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts.

Recombinant retroviruses that transduce the simian virus 40 (SV40) large T antigen or the polyomavirus large T antigen as well as encoding resistance to antibiotic G418 were used to investigate whether these genes alone were sufficient for immortalization of primary cells. The results provided definitive evidence that either viral gene can efficiently establish primary fibroblasts. The capability of the SV40 large T antigen to establish primary fibroblasts was undiminished by a mutation that alters its binding to sequences within the origin of replication. Surprisingly, most of the primary cells established by the expression of the SV40 large T antigen did not have a transformed phenotype. This suggests that transformation by SV40 is not simply due to a high level of expression of the SV40 large T antigen and stabilization of cellular p53.