The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.Genetic heterogeneity is one of the most remarkable and pervasive characteristics of established or immortal cell lines, especially those of high tumorigenicity (6,32,33,53). Whereas normal cells in vivo or in culture are predominantly or entirely diploid, tumors and immortal cell lines are invariably aneuploid. Such transformed cells exhibit frequent chromosomal alterations, in particular translocations (53) and gene amplifications (6, 37), some of which are associated with specific tumor types (6,24,53). They also undergo progressive dedifferentiative changes in gene expression, such as ectopic production of gene products (typically embryonal genes) which are not found in normal differentiated cells of that type (32), and clonal loss of gene products which would be expressed in normal cells of that type (33). These factors imply a fundamental shift toward genetic instability associated with transformation to immortality and tumorigenesis. Although homologous recombination is a likely common mechanism for DNA translocation and amplification (1, 10, 17) and a possible mechanism for alterations in gene expression (6, 24), recombination frequencies have not been directly compared between immortally transformed cell lines and normal diploid cells.Most carcinogens prove to be mutagenic when assayed in Salmonella His-reversion assays which are specific for point or frameshift mutations (6), but many are poorly detecte...

No abstract

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.