Transfer of immortality by transfection of genomic DNA from SV40 established cell lines into rat embryo fibroblasts

The cellular immortalization activity of cloned genes can be identified either in a colony-forming assa of transferred primary rat embryo fibroblasts or in a co-operation assay together with ras. However the demonstration of immortalization activities carried by cellular genes has not been reported. Here we establish that SV40 early genes integrated in genomic DNAs can be stably transferred into rat embryo fibroblasts and selected via their immortalization activity. Attempts to extend this assay to the identification of dominant genes putatively involved in the immortality of several other immortal post-crisis or tumor cells have been unsuccessful suggesting that the immortal phenotype can be brought about through different pathways.     

Transfer of immortality by transfection of genomic DNA from SV40 established cell lines into rat embryo fibroblasts

The cellular immortalization activity of cloned genes can be identified either in a colony-forming assa of transfected primary rat embryo fibroblasts or in a co-operation assay together with ras. However the demonstration of immortalization activities carried by cellular genes has not been reported. Here we establish that SV40 early genes integrated in genomic DNAs can be stably transferred into rat embryo fibroblasts and selected via their immortalization activity. Attempts to extend this assay to the identification of dominant genes putatively involved in the immortality of several other immortal post-crisis or tumor cells have been unsuccessful suggesting that the immortal phenotype can be brought about through different pathways.     

Characterization of human umbilical vein endothelial cell lines produced by transfection with the early region of SV40

Human umbilical vein endothelial cells were transfected by electroporation with the plasmid pSV3neo, containing the early region of simian virus 40. The resultant "cell lines" divide rapidly (population doubling time of 33 h) for up to 24 passages in medium supplemented with 5% (v/v) serum and 2.5 micrograms/ml endothelial cell growth supplement. Several of these lines express basal levels of ICAM-1 and MHC class I but not MHC class II. One cell line, designated SGHEC-7, retained a number of differentiated endothelial cell functions throughout its lifespan. These functions include increased production of tissue plasminogen activator in response to histamine, thrombin, and PMA. Stability of function and rapid growth over 24 passages endow these cells with a number of advantages over primary cultures. The homogeneous cell population and consistency of response make them ideal for biochemical and immunological studies hereto impractical with primary human endothelial cells. The success of this approach may allow the production of functional cell lines from other vascular beds.     

The immortalisation of rat hepatocytes by transfection with SV40 sequences

Abbreviations EGTA – ethylene bis(oxyethylenenitrilo)-tetraacetic acid; F12 – Ham's F12; FBS – foetal bovine serum; HBSS – Hank's balanced salt solution; HDM – hormonally defined medium; HEPES – 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid; NBS – new born calf serum; WME – Williams' medium E.     

Production of immortal human endothelial cell lines by strontium phosphate transfection and electroporation of SV40 sequences

Summary Eleven human endothelial cell lines have been produced by introducing sequences from the DNA tumor virus SV40 into human umbilical vein endothelial cells either by strontium phosphate coprecipitation or electroporation. The resultant lines were confirmed as being endothelial in origin by their production of endothelial-specific von Willebrand factor. The growth characteristics of the different lines in normal and reduced levels of serum was determined, as was their cellular response to endothelial cell growth supplement in combination with heparin, basic fibroblast growth factor, transforming growth factor-alpha, and epidermal growth factor.     

Optimization of electroporation for transfection of human fibroblast cell lines with origin-defective SV40 DNA: development of human transformed fibroblast cell lines with mucopolysaccharidoses (I-VII)

To simplify the process of transfection of human fibroblasts and to acquire a suitable number of transformants, we investigated experimental conditions of electric pulse-induced transfection of human fibroblasts using origin-defective simian virus 40 DNA (SV40 (ori-) DNA). Voltage, pulse duration, number of pulses and the concentration of SV40 (ori-) DNA led to the formation of 10 to 30 foci/25 cm2 6 weeks after transfection, using 2 to 3 x 10(6) cells and a square wave pulse generator. Optimal condition was determined to be 2 or 3 pulses at a voltage of 1500 to 2000 V/0.4 cm with 30 microseconds pulse width, using 2 micrograms of linearized SV40 (ori-) DNA. With this approach we developed human transformed fibroblasts cell lines with all types of mucopolysaccharidoses. The transformed fibroblasts grew rapidly and the saturation density exceeded that of the parental cells. All the transformed cell clones expressed T antigen, and deficiency in specific enzymes was conserved. A point mutation which occurred in the human beta-glucuronidase gene in a patient with mucopolysaccharidosis type VII was also conserved.     

Expression of SV40 tumor antigen in SV40 transformed teratocarcinoma-derived cell lines

The relative importance of viral tumor antigen expression and the cellular background in the maintenance of a transformation phenotype was examined in five SV40-transformed teratocarcinoma-derived cell lines. These cell lines show qualitative differences in growth characteristics associated with transformation, and vary in their state of differentiation. Viral T antigen expression was evaluated by two criteria: 1) the amount of immunoprecipitated antigen in growing cells, and 2) the amount and rate of antigen synthesis in density-inhibited cells. There was no direct correlation found between retention, or rate of synthesis, of the viral T antigen and the degree of transformation. These findings imply that the cellular environment has a more important influence on the growth properties of a stably transformed cell than the quantitative levels of viral T antigen expression.     

Species-specific functional interactions of DNA polymerase alpha-primase with simian virus 40 (SV40) T antigen require SV40 origin DNA.

Physical and functional interactions of simian virus 40 (SV40) and polyomavirus large-T antigens with DNA polymerase alpha-primase were analyzed to elucidate the molecular basis for the species specificity of polymerase alpha-primase in viral DNA replication. SV40 T antigen associated more efficiently with polymerase alpha-primase in crude human extracts than in mouse extracts, while polyomavirus T antigen interacted preferentially with polymerase alpha-primase in mouse extracts. The apparent species specificity of complex formation was not observed when purified polymerase alpha-primases were substituted for the crude extracts. Several functional interactions between T antigen and purified polymerase alpha-primase, including stimulation of primer synthesis and primer elongation on M13 DNA in the presence or absence of the single-stranded DNA binding protein RP-A, also proved to be independent of the species from which polymerase alpha-primase had been purified. However, the human DNA polymerase alpha-primase was specifically required for primosome assembly and primer synthesis on SV40 origin DNA in the presence of T antigen and RP-A.     

The crystal structure of the SV40 T-antigen origin binding domain in complex with DNA

DNA replication is initiated upon binding of “initiators” to origins of replication. In simian virus 40 (SV40), the core origin contains four pentanucleotide binding sites organized as pairs of inverted repeats. Here we describe the crystal structures of the origin binding domain (obd) of the SV40 large T-antigen (T-ag) both with and without a subfragment of origin-containing DNA. In the co-structure, two T-ag obds are oriented in a head-to-head fashion on the same face of the DNA, and each T-ag obd engages the major groove. Although the obds are very close to each other when bound to this DNA target, they do not contact one another. These data provide a high-resolution structural model that explains site-specific binding to the origin and suggests how these interactions help direct the oligomerization events that culminate in assembly of the helicase-active dodecameric complex of T-ag.     

Assembly of the replication initiation complex on SV40 origin DNA

The assembly of the complex that forms over the simian virus 40 origin to initiate DNA replication is not well understood. This complex is composed of the virus-coded T antigen and three cellular proteins, replication protein A (RPA), DNA polymerase α/primase (pol/prim) and topoisomerase I (topo I) in association with the origin. The order in which these various proteins bind to the DNA was investigated by performing binding assays using biotinylated origin DNA. We demonstrate that in the presence of all four proteins, pol/prim was essential to stabilize the initiation complex from the disruptive effects of topo I. At the optimal concentration of pol/prim, topo I and RPA bound efficiently to the complex, although pol/prim itself was not detected in significant amounts. At higher concentrations less topo I was recruited, suggesting that DNA polymerase is an important modulator of the binding of topo I. Topo I, in turn, appeared to be involved in recruiting RPA. RPA, in contrast, seemed to have little or no effect on the recruitment of the other proteins to the origin. These and other data suggested that pol/prim is the first cellular protein to interact with the double-hexameric T antigen bound to the origin. This is likely followed by topo I and then RPA, or perhaps by a complex of topo I and RPA. Stoichiometric analysis of the topo I and T antigen present in the complex suggested that two molecules of topo I are recruited per double hexamer. Finally, we demonstrate that DNA has a role in recruiting topo I to the origin.     

Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion

Incubation of 32P-5' end-labeled DNA fragments of less than 500 bp with excess amounts of the 3' leads to 5', double strand-dependent nuclease Exonuclease III generally results in single-stranded products of slightly more than half the size of the uncleaved substrate. When such restriction fragments of known size and sequence containing the lac operator were incubated with purified lac repressor, Exonuclease III cleavage was blocked at the 3' borders of the operator on each strand. It was possible to define the DNA sequence between the two boundaries of repressor-mediated exonuclease blockade by electrophoresing the single-stranded, protected products in urea-containing polyacrylamide gels in parallel with a dimethylsulfate modification-cleavage digest of the end-labeled, uncleaved substrate. The same approach was applied to an analysis of sites of large SV40 T antigen protection in the vicinity of the origin of SV40 DNA replication. Three discrete boundaries of apparent protection were observed--one on the "late" side of the origin and two on the "early" side. These sequences may constitute the 3' borders of discrete T antigen-binding sites in the origin region. Alternatively, one or more of these blockade points may signify regions of the genome which undergo conformational changes resulting in Exonuclease III resistance due to vicinal T antigen binding.     

The initiation of SV40 DNA synthesis is not unique to the replication origin

Replicative intermediates of SV40 were isolated, digested with the restriction endonuclease Bgl I and examined by electron microscopy. Over 98% of the replicative intermediates isolated following infection with wild-type virions at 33 degrees, 37 degrees or 40 degrees C or with tsA209 at 33 degrees C had initiated replication about 35 nucleotides to one side of the Bgl I site. Approximately 1% of the molecules had initiated replication about 2400 nucleotides from the Bgl I site. The remaining molecules may have initiated at other sites. When tsA209 virion-infected cultures were shifted to 40.5 degrees C for 90 min, the relative rate of thymidine incorporation into superhelical viral DNA dropped by more than 97%. The remaining incorporation was not due to "leakiness." The label incorporated into mature superhelical molecules during brief pulses was not preferentially incorporated near the terminus of replication as it was at 33 degrees C. Approximately 33% of the incorporated label represented repair synthesis. Electron microscopy revealed that half of the replicative intermediates formed under these conditions appear to have been initiated randomly around the SV40 genome. Rolling circle molecules contaminated all the preparations of replicative intermediates.     

Efficient immortalization and morphological transformation of human fibroblasts by transfection with SV40 DNA linked to a dominant marker

Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno-deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40-transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line.     

Complete enzymatic synthesis of DNA containing the SV40 origin of replication

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.     

Ultrastructural and cytochemical features of SV 40 transformed hypothalamic cell lines

Summary A continuous cell line was previously obtained by Simian Virus (SV) 40 transformation of primary cultures of dissociated mouse fetal hypothalami. One clone from this cell line has been previously shown to possess some of the ultrastructural features, immunological properties and synthesizing capacities of magnocellular hypothalamic neurons which secrete vasopressin and neurophysins. The present paper reports on the morphological characterization of 14 other clones or subclones of the original cell line, using the following criteria: phase contrast microscopy, electron microscopy, Gomori's aldehyde fuchsin staining, cytochemical detection of -glucuronidase, immunochemical staining with antisera against bovine neurophysin I, bovine neurophysin II, lys-vasopressin, oxytocin, LH-RH and TRH.The results allowed the conclusion that the clones as well as the subclones can be distributed into two groups: 1) neurosecretory neurons which all possess several of the ultrastructural and cytochemical features of the neurophysin-vasopressin synthesizing clone, and 2) primitive nerve cells which are devoid of such features but display numerous bundles of filaments. In addition some clones were found to display intermediate features between groups 1 and 2. A similar diversity was observed within clones of the original strain and subclones of a neurosecretory clone. It is suggested that the primitive clones could represent precursors of the neurosecretory clones.This work is dedicated to Professor W. Bargmann in honour of his 70th birthday