Integration and excision of SV40 DNA from the chromosome of a transformed cell

The single insertion of SV40 DNA present in the genome of the 14B line of transformed rat cells has been cloned in procaryotic vectors. Analysis of the clones reveals a complex arrangement of viral sequences in which a small tract of DNA is inverted with respect to the major insertion. The nucleotide sequences at the two junctions show sharp transitions between cellular and viral sequences. The sequences which flank the viral insertion have been used as probes to clone the corresponding genomic sequences from the DNA of untransformed rat cells. Analysis of the structure of these clones shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration. When 14B cells are fused with uninfected simian cells a heterogeneous set of low molecular weight superhelical DNAs containing viral sequences is generated. These have been cloned in procaryotic vectors and their structures have been analyzed. All of them span the origin of SV40 DNA replication and are colinear with various segments of the integrated viral DNA and its flanking sequences. The shorter molecules contain part of the integrated viral genome and cellular sequences from one side of the insertion. They were therefore generated by recombination between the viral DNA and its flanking cellular sequences. The longer molecules contain cellular sequences from both sides of the insertion as well as an entire copy of the integrated viral DNA. They were therefore generated by recombination between the flanking cellular sequences. These results argue strongly against the involvement of specific excision enzymes, and rather are discussed in terms of a model involving replication of the integrated viral DNA followed by recombination for release of integrated viral sequences.     

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.     

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.     

Association of SV40 DNA sequences with nuclear matrix in SV40 transformed hamster fibroblasts

Nuclear matrices were isolated by the high-salt, non-ionic detergent method from SV40-transformed hamster fibroblasts (TSV5 cell line), and from hamster tumours derived from these cells. DNA isolated from matrices and total nuclei was hybridized with nick-translated SV40 DNA. The enrichment of matrix DNA with SV40 DNA sequences was observed in all five experiments with matrix DNA of TSV5 cells but only in five out of nine matrix DNA isolated from tumour cells.     

Characterization of nude mouse skin fibroblast cell lines transformed by combined action of SV40 and 3-methylcholanthrene

Properties of transformed cell lines derived from secondary cultures of newborn NMRI nu/nu (nude) mouse skin fibroblasts by the sequential exposure of 3-methylcholanthrene and a DNA virus, SV40, were studied. Such transformants were compared to cells transformed by 3-methylcholanthrene or SV40 alone for the tumourigenicity, T-antigen expression, different in vitro growth characteristics and natural killer (NK) cell sensitivity. Despite a considerable variation within a group, the cell lines transformed by the combination treatment as a group were more tumourigenic than cell lines of other groups. In addition, the cell lines transformed by the combination treatment showed increased amounts of T-antigen as compared to cell lines transformed by SV40 alone. They also had, on an average, shorter population doubling time, higher cell saturation density, and a higher amount of DNA per cell than cell lines transformed by SV40 alone. Combination treatment cell lines (5 out of 8) grew in soft agar, whereas cell lines transformed by SV40 or 3-methylcholanthrene alone did not. In conclusion, the cell lines transformed by the combination treatment of 3-methylcholanthrene and SV40 had properties related to malignancy more often than cell lines transformed by SV40 or 3-methyl cholanthrene alone.     

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     

Cyclic AMP levels in temperature sensitive SV40 transformed cell lines

The cAMP levels of the 3T3 cell line and its transformed derivatives were determined. The level was found to be lower in transformed cell lines than in parental 3T3 and to decrease at confluence in all cell lines tested. SV40 transformed 3T3 cell lines which are temperature sensitive in growth control were found to have lower cAMP levels than 3T3, but these levels were not temperature dependent. Retransformation of these cell lines by murine sarcoma virus did not markedly affect their cAMP levels.     

Nucleotide sequence preference at rat liver and wheat germ type 1 DNA topoisomerase breakage sites in duplex SV40 DNA.

The site specificities of the type 1 DNA topoisomerases (topo 1) from rat liver and wheat germ were investigated. The nucleotide sequence at break sites on duplex SV40 DNA were determined for 245 wheat germ topo 1 sites and 223 rat liver topo 1 sites over a region of 1781 nucleotides. The enzymes from the two different sources show similar, but not identical patterns of DNA strand breakage. The sites occur frequently, but are not broken with equal probabilities. Major sites of breakage occur on the average every one to two turns of the helix, thus if sites of breakage accurately represent topo 1 sites of activity, the DNA sequence alone would appear to place few limits on the access of the enzyme to DNA. Sequences around the strongest sites for both enzymes show a bias in base composition for the four nucleotides immediately 5' to the break site (-4 to -1 positions), but no bias is observed 3' to the site of breakage. Consensus sequences for both enzymes were determined. Variations from the consensus sequence appear to affect the two enzymes differently and may account for the differences observed in the specificity of breakage.     

Stabilization of SV40 transformed human fibroblast cytoplasmic thymidine kinase by ATP

Summary Human fibroblast cytoplasmic thymidine kinase is stabilized by ATP. Sedimentation in sucrose gradients shows that in the presence of ATP, cytoplasmic thymidine kinase has a higher molecular weight (54 000) than in the absence of ATP (28 000). Removal of ATP by dialysis results in the loss of enzyme activity. The subsequent addition of ATP restores activity following a second order time course. These results are interpreted to indicate that in a human fibroblast cell line, transformed by SV₄₀ virus, cytoplasmic thymidine kinase is a dimer in the presence of ATP, but a less active monomer in its absence. Mitochondrial thymidine kinase from the same cell line is not affected by ATP.     

Chemical synthesis of tridecanucleoside dodecaphosphate sequence of SV40 DNA

The preparation, by the phosphotriester approach, of d[C-T-A-T-T-C-C-A-G-A-A-G-T] from one tetranucleoside triphosphate and three trinucleoside diphosphate blocks is described. The use of the o-dibromomethylbenzoyl (DBMB) protecting group in oligodeoxyribonucleotide synthesis is described for the first time. Internucleotide linkages are protected by o-chlorophenyl groups which are finally removed by treatment with the N1, N1, N3, N3-tetramethylguanidinium salt of syn-4-nitrobenzaldoxime. The first phosphorylation step (leading to phosphodiester intermediates) is carried out by treatment with o-chlorophenyl phosphorodi-(1,2,4-triazolide) followed by treatment with water and triethylamine. 1-Mesitylenesulphonyl-3-nitro-1,2,4-triazole (MSNT) is used as the activating agent in the second phosphorylation step in which 5'-protected mono- and di-nucleotides are condensed with nucleoside building blocks containing unprotected 3'-hydroxy functions.     

Sequence effect on alkali-sensitive sites in UV-irradiated SV40 DNA.

Ultraviolet light at 254 nm induces various kinds of DNA damage. We have located and quantified the pyrimidine (6-4) pyrimidone photoproducts along three hundred and forty two nucleotides of SV40 DNA. The level of photoproduct induction varies greatly according to the position on the DNA, but unlike what happens with pyrimidine dimers, the very adjacent nucleotides do not play a major role in the frequency of formation. A new alkali-sensitive site has been found on the ACA sequence after UV irradiation. This complex lesion is insensitive to the T4 endonuclease V and the E. coli photolyase, and may be involved with mutagenesis.     

Integration of SV40 DNA into the cell genome and viral mutagenesis

Integration of DNA of a temperature-sensitive SV40 mutant (tsA239) into the cell genome was studied. The viral A gene (the oncogene) encodes the tumour T antigen which is ts in the mutant and is devoid of mutagenic and transforming activity under non-permissive conditions (40 degrees C). Clones of Chinese hamster cells infected by tsA239 mutant were analysed. Those infected by wild-type SV40 served as controls. As shown by dot-hybridization, SV40 DNA was detected in cells of 14 out of 18 clones infected by tsA mutant and incubated at 40.5 degrees C, and in all 20 clones infected by tsA mutant and incubated under permissive conditions (33 degrees C), the difference between the two groups being insignificant (p greater than 0.05). By means of blot-hybridization it was established that viral DNA was integrated into the cell genome of all 12 clones analysed, belonging to the three experimental series: infection by tsA mutant, incubation at 40.5 and 33 degrees C, infection by wt SV40, incubation at 40.5 degrees C. The number of integration sites ranged from one to four in different clones. Integration of SV40 DNA in tandems was observed. The data presented allow to conclude that integration per se does not play a crucial role in determining the mutagenic and transforming effect of the virus. Obviously, what matters is the activity of viral oncogene product - the T antigen.     

Development and characterization of SV40 immortalized rat parotid acinar cell lines

Summary Rat parotid salivary gland acinar cells were transfected by CaPO₄ precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 30 clonal cell lines, 2 were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist (isoproterenol), vasoactive intestinal peptide prostaglandin E₁, and forskolin were effective activators of intracellular cyclic adenosine 3′:5′-cyclic monophosphate production. Phenylephrine, carbamylcholine, and UTP were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without affect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express the SV40 large T antigen. Electron microscopic evaluation documented moderate to high levels of cytodifferentiation with the maintenance of tripartite junctional complexes, cellular polarization, and presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 22 and 36 h, respectively.     

Development and characterization of SV40 immortalized rat submandibular acinar cell lines

Summary Rat submandibular salivary gland acinar cells were transfected by CaPO₄ precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 27 clonal cell lines, two were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist, isoproterenol, vasoactive intestinal peptide, and prostaglandin E₁ were effective activators of intracellular cyclic AMP production. Epinephrine, norepinephrine, phenylephrine, acetylcholine, and P_2U-purinoceptor agonists were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without effect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express glutamine/glutamic acid-rich proteins, a submandibular acinar cell specific secretory protein family. Electron microscopic evaluation documented the maintenance of tripartite junctional complexes, cellular polarization, and the presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 25 h.     

Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13

Summary Comparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.     

Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.Abbreviations I-SF immortalized human skin fibroblasts - T-SF transformed human skin fibroblasts - FBS fetal bovine serum     

Integration and transcription of group C human adenovirus sequences in the DNA of five lines of transformed rat cells

We have used the Southern blotting technique to analyze the integration patterns of human adenovirus sequences in the DNA of four rat cell lines, F17, 8617, T2C4 and F4, which were transformed by Ad-2 virus, and 5RK clone 6, which was transformed by Ad-5 HindIII-G fragment. We have also analyzed the Ad-specific messenger RNAs synthesized in these cell lines, in 293 cells (an Ad-5 transformed human cell line), and in Ad-2 early infected human KB cells, using the RNA geltransfer hybridization technique. We were interested in whether the Ad sequences are integrated, what the integration patterns are, whether the transforming region is present in an intact form, and whether the transforming region and other early regions are expressed at the mRNA level.Our results show that the integration patterns of Ad sequences range from simple to quite complex. Cells from line 8617 contain a single copy of right-end sequences flanked by left-end sequences. T2C4 cells have four different left-end sequences and two different right-end sequences. 5RK cells contain multiple different pieces of left-end sequences. In agreement with the results of Sambrook et al. (1979a,b), F17 cells contain a single copy of the left 17% of the genome, and F4 cells contain multiple copies of the right 5% of the genome fused to the left ~ 68% of the genome. The complete Ad genome is not present in any of the cell lines, and different regions may not be equimolar. There are no specific sites on the cellular or viral genome at which integration occurs. In 8617, F17 and F4 cells the Ad-2 sequences appear to be located close together on a single chromosome, suggesting that the Ad sequences in these cells arose from a single integration event. F17, 8617, T2C4, F4, and probably 5RK, cells all have an intact early region E1a (map position 1·3–4·6); F17, 8617, T2C4 and F4 cells also have E1b (m.p. 4·6–11·2) intact. E1a and E1b are the regions responsible for transformation. 8617 cells also have an intact early region E4 (m.p. 99-91·5) and T2C4 cells have an intact early region E3 (m.p. 76–86).Ad-2 early infected KB cells were shown to synthesize major E1a-specific mRNAs of 13 S, 12 S and 9 S, and major E1b-specific mRNAs of 22 S and 13 S. All the transformed cells synthesize the E1a 13 S and E1a 12 S mRNAs, and all cells except 5RK synthesize the E1b 22 S and E1b 13 S mRNAs. Early infected KB cells synthesize E3-specific mRNAs of 26 S, 24 S, 22 S, 19 S, 12 S and 9 S: T2C4 cells synthesize the major 22 S and 19 S RNA species, and possibly the less pronounced E3 mRNAs. Early infected cells and 8617 cells synthesize E4-specific mRNAs of 19 S, 17 S, 14 S, 12 S, 11 S, 9 S and 8 S. 8617 cells also synthesize E4 mRNAs of about 23 to 24 S and 21 S. F4 cells synthesize 24 S and 19 S hybrid mRNAs that contain both E4 and E1a sequences: these RNAs arise because F4 cells contain a portion of the E4 region fused to the left end (m.p. 0) of the genome.Our results, as well as those from other laboratories, are consistent with the idea that the transformed phenotype of Ad transformed cells is maintained by expression of Ad genes in E1a and E1b.