Three distinct effects of SV40 T-antigen gene transfection on cellular differentiation

SV40 large T-antigen-induced transformation has been reported to block differentiation, but the mechanism(s) of this effect has not been established. The results presented here show that stable transfection of the SV40 T-antigen gene, via the pSV3neo plasmid, has at least three distinct effects on 3T3T adipocyte differentiation. Cells first show a decreased ability to undergo predifferentiation growth arrest, which is a prerequisite for in vitro 3T3T adipocyte differentiation. However, if predifferentiation growth arrest is accomplished by use of stringent differentiation-inducing culture conditions, adipocyte differentiation can occur with high frequency. The pSV3neo-transfected cell clones also show other modifications of the adipocyte differentiation process, including changes in nonterminal (reversible) and terminal (irreversible) steps of adipocyte differentiation. When compared to nontransfected 3T3T cells, the cell clones containing pSV3neo require markedly reduced growth factor concentrations to restimulate proliferation of nonterminally differentiated adipocytes and the terminal step of differentiation is also blocked. These results suggest that integration of the T-antigen gene, through pSV3neo transfection, has multiple effects on the cellular mechanisms of differentiation. It does not block the differentiation process per se; rather it appears to make cells highly sensitive to proliferation signals, thereby making differentiation more difficult.     

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.     

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.     

Extension of lifespan of human T lymphocytes by transfection with SV40 large T antigen.

Lymphocytes have a finite and predictable proliferative life span in culture similar to that observed in fibroblasts. In general, the senescence of human fibroblasts is inevitable and irreversible, but their proliferative life span can be extended by certain DNA tumor virus oncogenes, such as the large T antigen of the SV40 virus. Here, we show that human T lymphocytes (HTL) can be stably transfected with SV40 large T and that expression of T antigen extended the life span of T cell cultures. PHA-stimulated HTL were transfected with pSV3neo, an expression vector containing the SV40 early region and the neomycin resistance gene. Transfectants were selected for neomycin (G418) resistance. Control HTL, either mock transfected or transfected with pSV2neo (containing the neomycin resistance gene only), ceased proliferation after about 17 population doublings. In contrast, HTL transfected with pSV3neo underwent more than 170 doublings. pSV3neo-transfected cells expressed SV40 large T RNA, detectable by in situ hybridization, and SV40 T antigen, detectable by immunofluorescence. Greater than 95% of the transfected cells were CD4 positive. These results clearly show that SV40 large T enables HTL to escape senescence. Transfection with SV40 large T may be a valuable method for obtaining long term human T cell lines for studies of both aging and immunology.     

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.     

High yield and stable transformation of the unicellular green alga Acetabularia by microinjection of SV40 DNA and pSV2neo

SV40 DNA and pSV2neo were microinjected into isolated nuclei of Acetabularia mediterranea. The injected nuclei were implanted into anucleate cell fragments of the same species. Such combinations not only survived but also formed progeny. The F₁, F₂ and F₃ generations of these combinations were analyzed. In the case of SV40-treated cells T-antigen was expressed and accumulated in the nuclei of all three generations studied as shown by indirect immunofluorescence. Nuclear exchange experiments revealed expression of the T-antigen only if a transformed nucleus but not if only a transformed cytoplasm was involved. Transformation by pSV2neo, a chimeric gene with a selectable marker was demonstrated by the induction of G-418 resistance as well as immunofluorescence. Genomic DNA was isolated from gametes, originating in cysts from the F₁, F₂ and F₃ generations of injected cells, and subjected to Southern analysis. These experiments demonstrated that both types of DNA are integrated into the host genome.     

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.     

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。     

Maintenance of expression of differentiated function of kidney cells following transformation by SV40 early region DNA

This study describes the isolation and characterization of epithelial cell lines that maintain their differentiated phenotype following the stable integration of SV40 genes. Epithelial cells were derived from a defined location of rabbit kidney, the thick ascending limb of Henle's loop, and were co-transfected with genes from the early region of SV40 together with pSV2-neo DNA (which confers resistance to the antibiotic G418). These cells were shown to be resistant to G418, express SV40 large T-antigen and continued to express differentiated characteristics typical of cells of their origin. Such characteristics include the expression of high levels of activity of both Na,K-ATPase and the functionally important Na,K,Cl-co-transport system, the synthesis of Tamm-Horsfall glycoprotein and the presence of a barium-sensitive K+ channel on the apical membrane surface.     

Transformation of human cultured fibroblasts with plasmids carrying dominant selection markers and immortalizing potential

The disadvantages of using human cultured cells for biochemical and genetic studies are their limited lifespan in vitro and their lack of chemical selection markers. These problems are now overcome by transfecting human cultured fibroblasts with the pSV3-gpt and pSV3-neo plasmid DNA which carry genes coding for the immortalizing SV40 large T-antigen and dominant selection markers. Transformed human fibroblasts were obtained at a frequency of about 10(-5) with both selection systems. These transformed cells showed a twofold increase in growth rate and three to tenfold increase in cell number at confluence. The improved growth characteristics were associated with the expression of the SV40 T-antigen detected with immunoprecipitation. These cell lines also changed from their usual spindle shapes to an epithelioid morphology characteristic of transformed cells. From 60 to 100% of the cells transfected with pSV3 plasmid DNA demonstrated numerical and structural abnormalities in their karyotypes. Cells transfected with DNA from a similar plasmid, pSV2-neo, which differed from the pSV3-neo plasmid only by missing the sequence encoding the complete early region of SV40, neither expressed T-antigen nor showed any change in morphology, improvement in growth characteristics or abnormalities in karyotype. However, they were still selectable with the aminoglycoside G-418. Therefore, by appropriate choice of vector plasmids, dominant selection markers and improved growth characteristics can be imparted separately or simultaneously to human fibroblasts. The morphological, biochemical and chromosomal changes resulting from such transformations must be recognized in using this approach for biochemical and genetic studies.     

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.     

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.     

DNA-mediated gene transfer in Friend leukemia cells by cotransfection of simian virus 40 DNA with herpes simplex virus thymidine kinase DNA.

Thymidine kinase-negative Friend leukemia cells were cotransfected with simian virus 40 (SV40) DNA and thymidine kinase gene DNA of herpes simplex virus type 1. The transfected thymidine kinase-positive cells were selected in HAT medium, and SV40 T-antigen expression was observed over many months in cells cultured under selective conditions, and after adaptation to normal growth medium under nonselective conditions. It was shown by Southern blot hybridization that SV40 DNA was integrated in multiple copies in the chromosomal DNA of several clones. All SV40 DNA-containing Friend leukemia cell clones analyzed were able to undergo induced erythroid differentiation. Induced cultures still expressed SV40 T-antigen to the same extent that untreated control cultures did.     

BHK-21-derived cell lines that produce basic fibroblast growth factor, but not parental BHK-21 cells, initiate neuronal differentiation of neural crest progenitors.

We present evidence that basic fibroblast growth factor (bFGF)-producing cells stimulate primary differentiation of neurons from neural crest progenitors. Baby hamster kidney (BHK-21) cells were stably cotransfected with plasmid pSV2/neo, which contains the gene conferring resistance to the neomycin analog G418 and expression vectors containing the human bFGF cDNA. Various clones, which differed in their bFGF production levels, were isolated. Homogeneous neural crest cells were cultured on monolayers of bFGF-producing, BHK-21-derived cell lines. While the parental BHK-21 cells, which do not produce detectable bFGF, had poor neurogenic ability, the various bFGF-producing clones promoted a 1.5- to 4-fold increase in neuronal cell number compared to the parental cells. This increase was correlated with the levels of bFGF produced by the different transfected clones, which ranged between 2.3 and 140 ng/mg protein. In contrast, no stimulation of neuronal differentiation was observed when neural crest cells were grown on monolayers of parental BHK cells transfected with plasmid pSV2/neo alone, or on a parental BHK-derived clone, which secretes high amounts of recombinant vascular endothelial growth factor (VEGF). Furthermore, the neuron-promoting ability of bFGF-producing cells could be mimicked by addition of exogenous bFGF to neural crest cells grown on the parental BHK line. A similar treatment of neural crest cells grown on laminin substrata, instead of BHK cells, resulted in increased survival of non-neuronal cells, but not of neurons (see also Kalcheim, C. 1989, Dev. Biol. 134, 1-10). Taken together, these results suggest that bFGF stimulates neuronal differentiation of neural crest cells by a cell-mediated signalling mechanism.     

Transformation of DNA repair-deficient human diploid fibroblasts with a simian virus 40 plasmid

Fibroblasts from patients with xeroderma pigmentosum (XP) complementation groups A, C, D, E, and G, as well as Bloom syndrome (BS) and Fanconi anemia (FA) have been transfected with a plasmid, pSV7, containing the early region of Simian virus 40 (SV40). All of the cultures exhibited cytologic changes characteristic of transformed cells and expressed T-antigen. They also contained integrated copies of DNA derived from the vector, and in several cases, extrachromosomally replicated DNA. Not all of the transfected cultures became immortalized. The transformed xeroderma pigmentosum (XP) cultures retained their UV-sensitive phenotype in all but one case. The BS and FA cell lines retained their characteristic phenotype. All of the cultures, except the BS cells, can be readily transfected with the plasmids, pSV2neo and pSV2gpt.     

Insulin-induced mitogenesis associated with transformation by the SV40 large T antigen.

Simian virus 40 (SV40) large T antigen-transformed cells typically show a markedly reduced serum requirement for growth and the inability to growth arrest and differentiate. An SV40 large T antigen-transformed 3T3 T cell line, CSV3-1, that can growth arrest and differentiate into adipocytes with high efficiency has, however, recently been described (Scott et al: Proc. Natl. Acad. Sci. U.S.A. 86:1652-1656, 1989; Estervig et al: J. Virol. 63:2718-2725, 1989; J. Cell. Physiol. 142:552-558, 1990). The results of the current studies using these cells show that whereas quiescent 3T3 T cells show no mitogenic response to insulin, quiescent CSV3-1 cells show a highly significant insulin-induced mitogenic responsiveness in the absence of other added growth factors. Maximum mitogenesis was observed at an insulin concentration of 1 microgram/ml, which induced 40-70% of the cells to undergo DNA synthesis within 48 hours. The half maximum response was achieved with 1-10 ng/ml of insulin. Insulin's mitogenic effect on CSV3-1 cells was evident under several different culture conditions that induce quiescence and was not mediated by any detectable autocrine growth factors that might make CSV3-1 cells competent to respond to insulin. In CSV3-1 cells insulin appears to act on its own receptor rather than on the IGF-1 receptor, because at comparable dosages IGF-1 is 10- to 100-fold less effective than insulin. Insulin also is shown to be a mitogen for another SV40-transformed cell line, CSV3-35, which can be growth arrested; in contrast insulin has no mitogenic effect on two control cell lines that are stably transfected with pSV2neo, a plasmid containing SV40 early promoter/enhancer but lacking large T antigen gene: These results suggest a significant relationship between SV40 T antigen-associated transformation and the expression of mitogenic responsiveness to insulin.