In the Ad12-transformed hamster cell line T637 with 15 copies of viral DNA inserted at a single chromosomal site, extensive alterations, mainly increases, in the levels of DNA methylation in the HpaII (5´-CCGG-3´) and HhaI (5´-GCGC-3´) sequences were apparent in the retrotransposon sequences of the about 900 copies of intracisternal A particle (IAPI) genomes [62]. The ~900 copies of IAPI sequences are a constitutive part of the hamster genome [115, 116]. In Ad12-transformed hamster cells, extensive changes in DNA methylation were also noted in the MHC class I and II, the Ig Cµ, the serine protease, and cytochrome P450 genes of the hamster cell genome. At least in the IAPI sequences, the increases in DNA methylation persisted in the revertant TR3 which had lost all 15 copies of Ad12 DNA [62]. Apparently, the alterations of the methylation patterns in the cellular genome are not dependent on the continued presence of the viral transgene DNA. The ~900 copies of IAPI DNA are distributed among many of the hamster chromosomes, often on their short arms [117, 118]. Since the increases in IAPI methylation were extensive, the effects of the Ad12 DNA insertion at a single chromosomal site had to transgress this site and lead to a disturbance in DNA trans-methylation patterns in the cellular genome even on different chromosomes.
Repetitive sequences in the mammalian genome appear to be particularly prone to respond with altered methylation patterns to perturbations in the genome caused by foreign DNA insertions. We surmise that the selection of the genes and DNA segments influenced in trans by the foreign DNA integration event might depend on the site of transgene insertion. The mechanism of this modulation of DNA methylation in the recipient genome remains unknown; it might be sought in the direct interaction of neighboring chromosomes. Soluble factors could obviously also have impacts. Further evidence in support of the contributions that foreign DNA insertions rendered in altering DNA methylation patterns in the recipient genome came from experiments in which we generated clonal hamster BHK21 cell lines with multiple copies of integrated bacteriophage lambda DNA. The integration phenomena of lambda DNA resembled those of Ad12 DNA in that multiple copies of the phage DNA came to reside at a single site of the hamster chromosome and became progressively de novo methylated. However, in contrast to the integrated Ad12 DNA, the integrated lambda DNA was not detectably transcribed. Alterations in cellular DNA methylation patterns were also observed in the IAPI sequences and could be unequivocally documented by the bisulfite genomic sequencing method with which 35 5´-CG-3´ dinucleotide positions were analyzed in a subsegment of the IAPI DNA region [119]. Even a transcriptionally inert transgene, like lambda DNA, had led to alterations in the methylation profiles in the IAP transposons.
The question arose whether such differences in methylation patterns among the different copies of the ~900 IAPI equivalents might have preexisted in different BHK21 cell clones. We therefore examined >70 individual BHK21 cell clones for differences in methylation patterns in the investigated IAPI segment both by HpaII and HhaI restriction patterns and by bisulfite genomic sequencing. Differences in patterns were not detectable [119]. Of course, we could not scrutinize thousands of individual cell clones for homogeneous methylation patterns. Nevertheless, on the basis of the available evidence, the preferred interpretation of a causative effect of foreign DNA integration on methylation patterns in trans will be pursued in future experimental projects.
We also entertained the possibility that the abortive infection of BHK21 cells with Ad12 [79, 120] with the transcription and expression of exclusively early Ad12 gene products [121] might have affected the stability of cellular DNA methylation patterns. At least on a time scale of days after Ad12 infection, changes in patterns of IAPI genome methylation could not be documented in BHK21 cells [119]. Productively Ad12-infected human or Ad2-infected hamster cells will also be examined for global changes in methylation patterns in the cellular genomes.
The method of methylation-sensitive representational difference analysis (MS-RDA) is based upon a subtractive hybridization protocol after selecting against DNA fragments, which were heavily methylated and, therefore, not cleaved by the HpaII restriction endonuclease [122]. We applied this method to the investigation of transcripts from bacteriophage lambda DNA-transgenic hamster cell lines in comparison to hamster cell lines devoid of integrated lambda DNA [123]. By using the suppressive hybridization technique for the analysis of cDNA preparations from non-transgenic, Ad12 DNA-transgenic and lambda DNA-transgenic hamster cells, several cellular genes were cloned which had altered transcriptional profiles in the transgenic as compared to the non-transgenic cells. Among individual non-transgenic hamster cell clones investigated as negative controls, no differences in cDNA isolates, and hence transcriptional profiles, were observed. We also studied these changes in one lambda DNA-transgenic mouse strain: hypermethylation was found for the imprinted IGF2R gene for DNA from heart muscle. Two mouse lines transgenic for an Ad2 promoter-indicator gene construct showed hypomethylation in the interleukin 10 and IGF2R genes. We concluded that in Ad12 DNA- or lambda DNA-transgenic hamster cells or mice, cellular methylation and transcription patterns can be critically altered [122]. Detailed investigations on the heterogeneity of DNA transcription patterns in about 1170 genes among individual clones of BHK21 and T637 cells have revealed only minimal differences in five of these genes by DNA array analyses between the two cell lines and among different clones of each cell line (N. Hochstein and W. Doerfler, unpublished experiments).
Since the insertion of foreign DNA into established mammalian genomes has become a preferred regimen in experimental biology, e.g., in the generation of transgenic organisms, and increasingly also in gene therapy, I consider it an important problem to pursue the unanticipated, likely unwanted, effect of foreign DNA integration on the stability of the recipient genome. Alterations of patterns of DNA methylation might be merely one, but an experimentally recognizable, manifestation of this disturbance [7]. These problems may have considerable relevance for certain regimens in gene therapy in which the fixation of foreign DNA in an established human genome is considered. When retroviral gene transfer vectors were used to chromosomally fix the human adenine deaminase gene in children with hereditary immunodeficiency, rare T cell leukemias developed. In these cases, I consider the insertion of foreign DNA as one of the decisive factors explaining this unfortunate outcome of a well-intended medical procedure.
Towards a working hypothesis on viral oncogenesis. Viral oncogenesis is frequently accompanied by the integration of the viral genome into the genome of the transformed cell. Integration of viral DNA is a conditio sine qua non for transformation [124] in cells transformed by adenoviruses, by SV40, polyoma virus, by the papilloma viruses, and notably by retroviruses. Integration is, of course, an important mode of chromosomal fixation and continued expression of the viral genome in the transformed cell. In retroviral replication, proviral integration is an essential step in the viral life cycle. Conventionally, major attention has been directed towards the function of the expressed viral gene products to explain the mechanism of viral oncogenesis. Having identified the viral “culprit” does not exclude the possibility that the real action is somewhere else, namely in its direct effect on the recipient genome. For some time, we have pursued the possibility that the alterations of DNA methylation patterns enacted in the wake of viral DNA insertion are a general phenomenon following the insertion of any foreign DNA [3-5, 7]. Altered methylation patterns then might be an indicator of more general perturbations in the cellular genome, which reach far beyond the immediate site of viral DNA integration. Furthermore, altered methylation patterns forebode changes in transcriptional patterns as well. Hence, upon foreign DNA insertion the recipient genome has undergone dramatic functional alterations, which might well be at the center of the oncogenic transformation process. Using the Ad12-hamster tumor system as a very efficient experimental model, we have only begun to document changes in cellular transcription patterns in Ad12-induced tumors [51].
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