Adenovirus type 12 (Adl2) is oncogenic in neonate hamsters (1, 2), and the viral genome can chromosomally integrate into the genome of hamster cells (3-7). Adl2 DNA insertion is not site-specific; transcriptionally active cellular DNA sequences seem to be preferred targets (8, 9). The molecular mechanism of Adl2 DNA insertional recombination is akin to nonhomologous recombination, as studied in a cell-free system (10, 11). We have pursued the possibility that foreign (Adl2) DNA insertion contributes to the process of tumorigenic transformation by Adl2. The conventional notion of insertional mutagenesis disrupting cellular DNA at the immediate locus of foreign DNA integration has been extended to the concept that cellular gene activity could be affected at many different cellular sites close to or remote from the locus offoreign DNA insertion due to changes in DNA methylation patterns (5, 6, 12).In Adl2-transformed hamster cells, in Adl2-induced hamster tumor cells, and in hamster cells carrying integrated Adl2The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5515 genomes, integrated plasmid, or bacteriophage A DNA without transformed phenotype, the methylation of several cellular DNA segments is markedly enhanced in comparison with BHK21 or primary hamster cells. The randomly selected hamster cellular DNA probes used in methylation analyses have been localized by fluorescent in situ hybridization (FISH) to different hamster chromosomes that do not carry Adl2 DNA. Thus, integration of foreign DNA in hamster cells exerts a distinct trans effect on the methylation of several cellular DNA segments located on different chromosomes.

The insertion of adenovirus type 12 (Ad12) DNA into the hamster genome and the transformation of these cells by Ad12 can lead to marked alterations in the levels of DNA methylation in several cellular genes and DNA segments. Since such alterations in DNA methylation patterns are likely to affect the transcription patterns of cellular genes, it is conceivable that these changes have played a role in the generation or the maintenance of the Ad12-transformed phenotype. We have now isolated clonal BHK21 hamster cell lines that carry in their genomes bacteriophage λ and plasmid pSV2neo DNAs in an integrated state. Most of these cell lines contain one or multiple copies of integrated λ DNA, which often colocalize with the pSV2neo DNA, usually in a single chromosomal site as determined by the fluorescent in situ hybridization technique. In different cell lines, the loci of foreign DNA insertion are different. The inserted bacteriophage λ DNA frequently becomes de novo methylated. In some of the thus-generated hamster cell lines, the levels of DNA methylation in the retrotransposon genomes of the endogenous intracisternal A particles (IAP) are increased in comparison to those in the non-λ-DNA-transgenic BHK21 cell lines. These changes in the methylation patterns of the IAP subclone I (IAPI) segment have been documented by restriction analyses with methylation-sensitive restriction endonucleases followed by Southern transfer hybridization and phosphorimager quantitation. The results of genomic sequencing experiments using the bisulfite protocol yielded additional evidence for alterations in the patterns of DNA methylation in selected segments of the IAPI sequences. In these experiments, the nucleotide sequences in >330 PCR-generated cloned DNA molecules were determined. Upon prolonged cultivation of cell lines with altered cellular methylation patterns, these differences became less apparent, perhaps due to counterselection of the transgenic cells. The possibility existed that the hamster BHK21 cell genomes represent mosaics with respect to DNA methylation in the IAPI segment. Hence, some of the cells with the patterns observed after λ DNA integration might have existed prior to λ DNA integration and been selected by chance. A total of 66 individual BHK21 cell clones from the BHK21 cell stock have been recloned up to three times, and the DNAs of these cell populations have been analyzed for differences in IAPI methylation patterns. None have been found. These patterns are identical among the individual BHK21 cell clones and identical to the patterns of the originally used BHK21 cell line. Similar results have been obtained with nine clones isolated from BHK21 cells mock transfected by the Ca2+-phosphate precipitation procedure with DNA omitted from the transfection mixture. In four clonal sublines of nontransgenic control BHK21 cells, genomic sequencing of 335 PCR-generated clones by the bisulfite protocol revealed 5′-CG-3′ methylation levels in the IAPI segment that were comparable to those in the uncloned BHK21 cell line. We conclude that the observed changes in the DNA methylation patterns in BHK21 cells with integrated λ DNA are unlikely to preexist or to be caused by the transfection procedure. Our data support the interpretation that the insertion of foreign DNA into a preexisting mammalian genome can alter the cellular patterns of DNA methylation, perhaps via changes in chromatin structure. The cellular sites affected by and the extent of these changes could depend on the site and size of foreign DNA insertion.

No abstract

We have been interested in the consequences of foreign DNA insertion into established mammalian genomes and have initially studied this problem in adenovirus type 12 (Ad12)-transformed cells or in Ad12-induced hamster tumors. Since integrates are frequently methylated de novo, it appears that they might be modified by an ancient defense mechanism against foreign DNA. In cells transgenic for the DNA of Ad12 or for the DNA of bacteriophage

No abstract