Spontaneous rearrangement of integrated simian virus 40 DNA in nine transformed rodent cell lines.

Frequencies of spontaneous DNA rearrangement within or near integrated simian virus 40 (SV40) DNA were measured in four transformed mouse and rat cell lines of independent origin and in five clones of the SV40-transformed mouse line SVT2. Rearrangements were detected as polymorphisms of restriction enzyme fragment length in subclones of the lines. At least 17% of the subclones of each line had detectable rearrangements. The rate of rearrangement was calculated to be at least 5 x 10(-3) events per cell per division. No rearrangements were detected in sequences of an immunoglobulin gene, part of the coding region of the mouse protein p53, and five proto-oncogenes. The possible role of recombination between duplicated segments of integrated SV40 DNA in generating rearrangements was studied in the five SVT2 clones, which differed in the number of duplications within a single SV40 DNA segment. The SVT2 clone that had no duplications, M3, became rearranged further at least as frequently as did closely related lines with one, two, or three duplications. Another line in this group that had one small duplication, X1, had a much higher frequency of rearrangement than did the others; integrated SV40 DNA of X1 became mostly rearranged within 100 cell divisions. The examples of M3 and X1 suggested that the high rate of rearrangement characteristic of integrated SV40 DNA was influenced more by the presence of particular sequences within or near integrated SV40 DNA than by the number or extent of duplicated sequences.     

Events preceding stable integration of SV40 genomes in a human cell line

We have examined the organization of integrated SV40 sequences in an uncloned population of a transformed human fibroblast cell line. Somatic cell hybrids between mouse B82 cells and human GM847 cells were examined for SV40 T-antigen expression and individual human chromosome presence. This analysis revealed that a functional SV40 genome is located on human chromosome 7. Restriction endonuclease digestion followed by blot hybridization of the parental human cell line revealed that it contains multiple normal and defective SV40 copies integrated into the host genome in tandem. A similar analysis of several T-ag+ hybrid cell lines indicated that the integrated viral sequences in different hybrid cell lines (thus in different cells of the original population) are very closely related but not always identical. Analysis of subclones of GM847 also revealed such differences. Based upon these results, we postulate that following the initial integration event, viral as well as the flanking host DNA sequences become unstable and are subject to deletions and rearrangements. This short-lived structural instability is followed by highly stable integration of SV40 which is maintained in these cells or their hybrid derivatives for at least hundreds of cell generations.     

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.     

Reacquisition of a functional early region by a mouse transformant containing only defective simian virus 40 DNA.

Viral DNA in simian virus 40-transformed mouse cells is capable of rearranging with passage. In this report, we show that such rearrangement can include an alteration in viral protein expression. SVT2, a simian virus 40-transformed mouse BALB/c 3T3 cell line, synthesizes only a super T antigen of molecular weight 100,000 without synthesizing the lytic-size large T or small t antigens with molecular weights of 94,000 and 17,000, respectively. Analyses of the integrated viral DNA revealed an early region of 4.4 kilobases instead of the lytic-size 2.7 kilobases. However, upon subcloning in either plastic or agarose or after being in culture for several passages, the appearance of lytic-size large T and small t antigens was detected. Concurrently, an early region of 2.7 kilobases, in addition to one of 4.4 kilobases, was observed.     

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

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

BK virus-transformed inbred hamster brain cells: status of viral DNA in subclones.

We have recently reported that viral DNA sequences in inbred LSH hamster brain cells transformed by the GS variant of BK virus (LSH-BR-BK) are present predominantly in a free form (Beth et al., J. Virol. 40:276-284, 1981). In this report, we confirm that the presence of viral DNA sequences in these cells is not due to virus production, since viral capsid proteins were not detected by immunoprecipitation. Furthermore, we examined the status of viral DNA in 15 subclones of this cell line and detected free and integrated viral DNA sequences in only 5 of the subclones. The other 10 subclones contained exclusively integrated viral DNA sequences, as shown by the blot hybridization of high-molecular-weight cell DNA which was uncleaved or digested with HincII, for which there are no sites in viral DNA. The arrangement of viral DNA in these clones was further analyzed by cleavage of cellular DNA with HpaII and HindIII. Mitomycin (0.03 microgram/ml) treatment of subclones containing only integrated sequences resulted in the appearance of free viral DNA sequences in some of these cells. This result supports the postulation that free viral DNA in LSH-BR-BK cells is made up of excision products of observed tandemly repeated integrated sequences. In addition to the large T- and small t-antigens, LSH-BR-BK and all of its 15 subclones contained two antigen species which were larger than large T and one species which was smaller than small t. The number of tumor antigens in the LSH- BR-BK cell line and its subclones with a large copy number in a free form was not more than in the subclones with low copy number and integrated DNA. This suggests that free viral DNA is not a template for tumor antigen production in transformed cells.     

Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants.

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.     

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.     

Chromosomal recombination and breakage associated with instability in mouse centrometric satellite DNA

A mouse L cell line containing the centromeric insertion of herpes thymidine kinase genes (tk) was previously shown to undergo a high frequency of DNA rearrangement at the site of tk insertion. Analysis of TK- revertants had demonstrated that DNA rearrangements were usually associated with DNA deletion and were always mediated by intrachromosomal recombinations. In this study, we further analyzed several TK+ subclones to examine the mode of DNA rearrangements in the absence of negative selection pressure. In two clones, LC2-3F and LC2-3E17, rearrangements were accompanied by DNA amplification and were mediated by intrachromosomal recombination. In subclone LC2-3E17-19, we further detected perturbations in the pattern of centromeric heterochromatization. This was associated with chromosome instability, as evidenced by chromosome breakage at the centromere. The analysis of three other sibling clones, LC2-3, LC2-6 and LC2-15, further suggests that reciprocal recombination events may play a role in such centromeric rearrangements. These results suggest that DNA rearrangements in the centromere may be mediated by a number of different mechanisms, and generally do not affect chromosome stability except when accompanied by changes in the pattern of heterochromatization.     

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.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

Karyotype evolution of the human HBL-100 cell line and mapping of the integration site of SV40 DNA

Cytogenetic analysis of the human HBL-100 cell line, that we have previously shown to harbour SV40 genetic information (Caron de Fromentel et al., 1985), reveals numerous chromosomal rearrangements as soon as the 30th in vitro passage. The karyotype is relatively stable during in vitro maintenance and even at late passages (approximately 70) when the cells have acquired the capacity to form tumors in nude mice. In all the somatic cell hybrids obtained after fusion of mouse 3T3-4E cells with HBL-100 cells, several human chromosomes are maintained and a derivative from chromosome 15-der(15)- is the most frequently observed. The der(15) marker is present in the HBL-100 cell line at every passage studied as well as in different cell lines derived from tumors induced by HBL-100 cells. The various hybrids, originally isolated for a transformed phenotype on the basis of their ability to grow in soft-agar, were all found to express the SV40 T-antigen. In situ hybridization of an SV40 DNA probe to chromosome spreads obtained from one of these hybrids shows that the integration site of the viral genome is located on the der(15) marker chromosome, at band 15q24. The possible cooperation of SV40 T-antigen with some other oncogene(s), required by human HBL-100 cells in order to express a malignant phenotype, is discussed.     

DNA binding properties of a mutant T antigen from the simian virus 40-transformed human cell line simian virus 80

We investigated whether the T antigen of the simian virus 40-transformed human cell line simian virus 80 ( SV80 ) specifically recognizes DNA sequences of its own template, i.e, the viral sequences integrated in the SV80 cellular genome. In vitro DNA binding experiments clearly indicated that, in contrast to wild-type T antigen, SV80 T antigen does not specifically bind to sites on the integrated viral DNA in SV80 cells.     

Simian virus 40 large T antigen oligomers: analysis of electrophoresis in the absence of detergent.

Large T antigen of simian virus 40 is found as monomeric and oligomeric species in transformed cells. These can be demonstrated in cell extracts by velocity centrifugation in sucrose gradients. We analyzed them further in a transformed human line cell (SV80) and a transformed mouse line cell (SVT2). Individual fractions from sucrose gradients were subjected to polyacrylamide gel electrophoresis in the absence of detergent. T-antigen species were then detected by protein blotting and antibody overlay with polyclonal anti-D2 T antibody or monoclonal Pab419, Pab101, or Pb1700 antibody. The rapidly sedimenting species (14S and larger) of large T antigen from both cell lines reproducibly showed two major bands with estimated molecular weights of 670,000 and 850,000. A third band of 1,200,000 was more prominent in SVT2 cells than in SV80 cells. In SV80 cells the slowly sedimenting species of large T antigen (5S to 11S) contained two reproducible bands. A band with a molecular weight of 95,000 was the predominant one in all fractions between 5S and 11S. A relatively minor band with a molecular weight of 230,000 was found in fractions between 9S and 11S. The low-molecular-weight forms were seen in SVT2 cells only when a prominent peak at 5S to 7S was present, that is, when extracts were stored before analysis. In fresh extracts, the low-molecular-weight bands and slowly sedimenting forms were absent.     

Integrated Simian Virus 40 DNA: Nucleotide Sequences at Cell-Virus Recombinant Junctions

DNA fragments containing the integrated viral DNA present in the simian virus 40 (SV40)-transformed rat cell lines SVRE9 and SVRE17 were cloned in procaryotic vectors, and the DNA sequences linking SV40 and cell DNA were determined. Comparison of the DNA sequences at the SV40-cell junctions in SVRE9 and SVRE17 cells with those of a previously characterized viral insertion from SV14B cells shows that no specific viral or cellular sequences occur at SV40-cell junctions and that the cellular DNA sequences adjacent to integrated SV40 DNA do not display the direct repeat structure characteristic of transposons and retrovirus proviruses.     

Structure of integrated simian virus 40 DNA in transformed mouse cells

The structure of integrated viral DNA sequences in four lines of simian virus 40 (SV40)-transformed Balb/c 3T3 cells has been probed using restriction endonucleases and the Southern (1975) transfer method. By considering data from a large number of restriction digests of DNA from each line, and by using a novel method of handling the data, we have constructed fairly detailed physical maps of the integrated DNA in each line. The most striking of the features of the maps described here is that none is easily explained by the integration of a single SV40 genome into the DNA of the host cell. Three of the lines contain at least two distinct integrated segments and the fourth contains a single segment longer than the viral DNA. Considered individually, only two of the seven segments that we have mapped might be unit length. Of the remaining five, two are longer and three are shorter than the viral genome. It seems likely, therefore, that at least in SV40-transformed Balb/c 3T3 cells simple, single integrations are rare.The endpoints of these seven segments of integrated DNA fall at many positions distributed over the entire genome, confirming earlier studies (Ketner &; Kelly, 1976; Botchan et al., 1976), which indicated that SV40 integration is not absolutely site-specific.Finally, one of the lines mapped here (SVB209) does not possess an intact SV40 early region, an observation that suggests the possibility that a normal viral large T polypeptide is not synthesized by this line.