Loss of viral genomes from hamster tumor cells and nonrandom alterations in patterns of methylation of integrated adenovirus type 12 DNA.

The insertion stability and DNA methylation patterns of integrated adenovirus type 12 (Ad12) genomes were investigated in Ad12-induced tumors and in tumor cell lines established from them as a function of time of passage under culture conditions. Upon subcultivation of cells from some of the tumors, the viral genomes were eliminated, apparently in a stepwise process with segments of the left termini of Ad12 DNAs persisting the longest. Morphological variants of these tumor cells lost all viral DNA and yet retained the oncogenic phenotype. All 13 independently isolated clones from one revertant line were devoid of Ad12 DNA. It could not be ruled out that very short sequence elements of viral DNA, such as promoters or enhancing sequences, could have persisted in these variants. The extent of viral DNA methylation was minimal in Ad12-induced tumors, although the viral genome was not extensively expressed, if at all. Upon passage in culture, the levels of viral DNA methylation increased. It was interesting that establishment of the final methylation pattern of integrated Ad12 DNAs required many cell generations after the fixation of foreign DNA in the host genome. The shift in methylation was nonrandom. The late parts of the inserted viral genomes became methylated more extensively than did the early gene segments.     

Fate of adenovirus type 12 genomes in nonpermissive cells

The fate of ³H-thymidine-labeled adenovirus type 12 deoxyribonucleic acid (DNA) was studied in Nil-2 cells of Syrian hamster origin. It was found that a substantial fraction of ³H-adenovirus type 12 DNA became degraded within 24 hr after infection and was released into the culture fluid. After infection of 5-bromodeoxyuridine (BUdR)-prelabeled cells with ³H-adenovirus type 12, viral DNA became readily separable from cellular DNA by equilibrium centrifugation in CsCl. Part of the viral radioactivity was found to shift gradually to the position of cellular DNA as time progressed after infection. When exponentially growing cells were exposed simultaneously to BUdR, 5-fluorodeoxyuridine, and ³H-adenovirus type 12, up to 50% of the viral radioactivity shifted within 24 hr from the density of viral DNA to that of cellular DNA after equilibrium centrifugation in CsCl. Upon denaturation of the cellular DNA, the isotope was preferentially found to be associated with the “heavy” strand which was synthesized after infection. Upon hybridization of the “heavy” and the “light” strands with sonically treated, denatured ³H-adenovirus type 12 DNA, small and nearly equal amounts of counts hybridized with both strands. The number of counts annealed was in a range similar to that of those annealed with the same amount of DNA derived from adenovirus type 12-transformed hamster cells. These results demonstrate that (i) a substantial proportion of the adsorbed virus becomes degraded within 24 hr; (ii) part of the degradation products is reutilized for cellular DNA synthesis; (iii) only a small fraction, mainly fragments, of viral DNA becomes integrated into both the newly synthesized and the parental strands of cellular DNA.     

An overview of the analysis of DNA methylation in mammalian genomes

DNA methylation at position C5 of the pyrimidine ring of cytosine in mammalian genomes has received a great deal of research interest due to its importance in many biological phenomena. It is associated with events such as epigenetic gene silencing and the maintenance of genome integrity. Aberrant DNA methylation, particularly that of chromosomal regions called CpG islands, is an important step in carcinogenesis. In order to elucidate methylation profiling of complex genomes, various methods have been developed. Many of these methods are based on the differential reactivity of cytosine and 5-methylcytosine to various chemicals. The combined use of these chemical reactions and other preexisting methods has enabled the discrimination of cytosine and 5-methylcytosine in complex genomes. The use of proteins that preferentially bind to methylated DNA has also successfully been used to discriminate between methylated and unmethylated sites. The chemical and structural dissection of the in vivo processes of enzymatic methylation and the binding of methyl-CpG binding proteins provides evidence for the complex mechanisms that nature has acquired. In this review we summarize the methods available for the discrimination between cytosine and 5-methylcytosine in complex genomes.     

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.     

New insights into stability of recombinant adenovirus vector genomes in mammalian cells

Recombinant adenoviruses are widely used in basic virology research, therapeutic applications, vaccination studies or simply as a tool for genetic manipulation of eukaryotic cells. Dependent on the application, transient or stable maintenance of the adenoviral genome and transgene expression are required. The newest generation of recombinant adenoviral vectors is represented by high-capacity adenoviral vectors (HC-AdVs) which lack all viral coding sequences. HC-AdVs were shown to result in long-term persistence of transgene expression and phenotypic correction in small and large animal models with negligible toxicity.Although there is evidence that adenoviral vectors predominantly persist as episomal DNA molecules with a low integration frequency into the host genome, detailed information about the nuclear fate and the molecular status of the HC-AdV genome once inside the nucleus is lacking. In recent years we have focused on analyzing and modifying the nuclear fate of HC-AdVs after infection of mammalian cells. We have focused on investigating the molecular DNA forms of HC-AdV genomes and we have designed strategies to excise and stably integrate a transgene from an episomal adenovirus vector genome into the host chromosomes by recombinases. This review article provides a state-of-the art overview of the current knowledge of episomal HC-AdV persistence and it discusses strategies for changing the nuclear fate of a transgene inserted into the HC-AdV genome by somatic integration into host chromosomes.     

Intracellular forms of adenovirus DNA. V. Viral DNA sequences in hamster cells abortively infected and transformed with human adenovirus type 12.

The persistence of viral DNA in BHK-21 cells abortively infected with human adenovirus type 12 has been investigated using reassociation kinetics. No indication of an increase in the amount of viral DNA per cell has been found. On the contrary, the amount of intracellular viral DNA sequences decreases rapidly after infection. Thus, free adenovirus type 12 DNA does not replicate in BHK-21 cells. The influence of the multiplicity of infection on the amount of persisting adenovirus type 12 DNA has also been explored. The viral DNA sequences persisting in four lines of hamster cells transformed in vitro by adenovirus type 12 at various multiplicities of infection have been quantitated and mapped by reassociation kinetics experiments using restriction endonuclease fragments of 3H-labeled adenovirus type 12 DNA. All the EcoRI restriction nuclease fragments of the adenovirus type 12 genome are represented in each of the four cell lines. Individual fragments of the viral genome are represented in multiple copies in non-equimolar amounts.     

Transforming DNA sequences in rat cells transformed by DNA fragments of highly oncogenic human adenovirus type 12.

Rat cell lines tranformed by viral DNA fragments, EcoRI-C and HindIII-G, of adenovirus type 12 DNA were analyzed for the viral transforming DNA sequences present in cell DNAs. Cell lines transformed by the EcoRI-C fragment of adenovirus type 12 DNA (leftmost 16.5% of the viral genome) contain most of the HindIII-G sequences of the HindIII-G fragment, but at a different frequency depending on the portions of the fragment. The sequence of the AccI-H fragment of adenovirus type 12 DNA (the left part of the HindIII-G; leftmost 4.5% of the viral genome) was detected dominantly in cells transformed by the HindIII-G fragment Southern blot analysis showed that viral DNA sequences are present at multiple integration sites in high-molecular-weight cell DNA from cells transformed by the EcoRI-C or HindIII-G fragment of adenovirus type 12 DNA. These results suggest that most of the HindIII-G sequences in cells transformed by the HindIII-G fragment are present as fragmented forms.     

Patterns of integration of viral dna sequences in the genomes of adenovirus type 12-transformed hamster cells

The patterns of integration of the viral genome have been analyzed in four hamster cell lines transformed by adenovirus type 12 (Ad12). It has previously been shown that in each of the cell lines HA12/7, T637, A2497-2 and A2497-3, the viral genome persists in multiple copies, and that different parts of the viral DNA are represented non-stoichiometrically (Fanning and Doerfler, 1976). All four cell lines are oncogenic when injected into hamsters.The DNA from each of the cell lines was extracted and cleaved in different experiments with restriction endonucleases Bam HI, Bgl II, Eco RI, Hind III, Hpa II or Sma I. The DNA fragments were separated on 1% agarose slab gels and transferred to nitrocellulose filters by the Southern technique. Ad12 DNA sequences were detected by hybridization to Ad12 DNA, which was ³²P-labeled by nick translation, and by subsequent autoradiography. In some experiments, the ³²P-labeled Eco RI restriction endonuclease fragments of Ad12 DNA were used to investigate the distribution of specific segments of the viral genome in the cellular DNA.For each cell line, a distinct and specific pattern of integrated viral DNA sequences is observed for each of the restriction endonucleases used. Moreover, viral sequences complementary to the isolated Eco RI restriction endonuclease fragments are also distributed in patterns specific for each cell line. There are striking differences in integration patterns among the four different lines; there are also similarities. Because the organization of cellular genes in virus-transformed as compared to normal cells has not yet been determined, conclusions about the existence or absence of specific integration sites for adenovirus DNA appear premature. Analysis of the integration patterns of Ad12 DNA in the four hamster lines investigated reveals that some of the viral DNA molecules are fragmented prior to or during integration. Analysis with specific restriction endonuclease fragments demonstrates that the Eco RI B, D and E fragments, comprising a contiguous segment from 0.17–0.62 fractional length units of the viral DNA, remain intact during integration in a portion of the viral DNA molecules. Although each cell line carries multiple copies of Ad12 DNA, the viral DNA sequences are concentrated in a small number of distinct size classes of fragments. This finding is compatible with, but does not prove, the notion that at least a portion of the viral DNA sequences is integrated into repetitive sequences, or else that the integrated viral sequences have been amplified after integration.In the three cell lines which were tested, the integration pattern is stable over many generations, with continuous passage-twice weekly-of cells for 6–7 months. In the three cell lines which were examined, the integration pattern is identical in a number of randomly isolated clones. Hence it can be concluded that the patterns of integration are identical among all cells in a population of a given line of transformed cells.     

Patch homologies and the integration of adenovirus DNA in mammalian cells.

The hamster cell line HE5 has been derived from primary hamster embryo cells by transformation with human adenovirus type 2 (Ad2). Each cell contains 2-3 copies of Ad2 DNA inserted into host DNA at apparently identical sites. The site of the junction between the right terminus of Ad2 DNA and hamster cell DNA was cloned and sequenced. The eight [corrected] right terminal nucleotides of Ad2 DNA were deleted. The unoccupied cellular DNA sequence in cell line HE5 , corresponding to the site of the junction between Ad2 and hamster cell DNA, was also cloned; 120-130 nucleotides in the cellular DNA were found to be identical to the cellular DNA sequence in the cloned junction DNA fragment, up to the site of the junction. The unoccupied and the occupied cellular DNAs and the adjacent viral DNA exhibited a few short nucleotide homologies. Patch homologies ranging in length from dodeca - to octanucleotides were detected by computer analyses at locations more remote from the junction site. When the right terminal nucleotide sequence of Ad2 DNA was matched to randomly selected sequences of 401 nucleotides from vertebrate or prokaryotic DNA, similar homologies were observed. It is likely that foreign (viral) DNA can be inserted via short sequence homologies at many different sites of cellular DNA.     

Variable effects of DNA-synthesis inhibitors upon DNA methylation in mammalian cells.

Post-synthetic enzymatic hypermethylation of DNA was induced in hamster fibrosarcoma cells by the DNA synthesis inhibitors cytosine arabinoside, hydroxyurea and aphidicolin. This effect required direct inhibition of DNA polymerase alpha or reduction in deoxynucleotide pools and was not specific to a single cell type. At equivalently reduced levels of DNA synthesis, neither cycloheximide, actinomycin D nor serum deprivation affected DNA methylation in this way. The topoisomerase inhibitors nalidixic acid and novobiocin caused significant hypomethylation indicating that increased 5-mCyt content was not a necessary consequence of DNA synthesis inhibition. The induced hypermethylation occurred predominantly in that fraction of the DNA synthesized in the presence of inhibitor; was stable in the absence of drug; was most prominent in low molecular weight DNA representing sites of initiated but incomplete DNA synthesis; and occurred primarily within CpG dinucleotides, although other dinucleotides were overmethylated as well. Drug-induced CpG hypermethylation may be capable of silencing genes, an effect which may be relevant to the aberrantly expressed genes characteristic of neoplastic cells.     

Persistence or loss of preimposed methylation patterns and de novo methylation of foreign DNA integrated in transgenic mice.

In cultured mammalian cells, foreign DNA can be integrated into the host genome. Foreign DNA is frequently de novo methylated in specific patterns with successive cell generations. The sequence-specific methylation of promoter sequences in integrated foreign DNA is associated with the long-term inactivation of eukaryotic genes. We have now extended these experiments to studies on transgenic mice. As in previous work, a construct (pAd2E2AL-CAT) has been used which consists of the late E2A promoter of adenovirus type 2 (Ad2) DNA fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT). This construct has been integrated in the non-methylated in the 5'-CCGG-3' premethylated form in the genomes of transgenic mice. DNA from various organs was analyzed by HpaII/MspI cleavage to assess the state of methylation in 5'-CCGG-3' sequences. The results demonstrate that the transgenic construct is in general stable. Non-methylated constructs have remained partly non-methylated for four generations or can become de novo methylated at all or most 5'-CCGG-3' sequences in the founder animal. Preimposed patterns of 5'-CCGG-3' methylation have been preserved for up to four generations beyond the founder animal. In the testes of two different founder animals and two F1 males, the transgenic DNA has become demethylated by an unknown mechanism. In all other organs, the transgenic DNA preserves the preimposed 5'-CCGG-3' methylation pattern. In the experiments performed so far we have not observed differences in the transmission of methylation patterns depending on whether the transgene has been maternally or paternally inherited. The 5'-CCGG-3' premethylated transgene does not catalyze CAT activity in several organs, except in one example of the testes of an animal in which the transgenic construct has become demethylated. In contrast, when the nonmethylated construct has been integrated and remained largely non-methylated, CAT activity has been detected in extracts from some of the organs.     

Gene silencing and endogenous DNA methylation in mammalian cells

It is known that transformed mammalian cells can spontaneously inactivate genes at low frequency by the de novo methylation of promoter sequences. It is usually assumed that this is due to DNA methyl transferase activity, but an alternative possibiity is that 5-methyldCTP is present in these cells and can be directly incorporated into DNA. The ongoing repair of DNA containing 5-methylcytosine will produce 5-methyldeoxycytidine monophosphate (5-methyldCMP), so the question arises whether this can be phosphorylated to 5-methyldCTP. We have tested this using three strains of CHO cells with different levels of 5-methyldCMP deaminase activity. That with the lowest enzyme activity, designated HAM⁻, has previously been shown to incorporate tritium labelled 5-methyldeoxycytidine into 5-methylcytosine in DNA, with a greater amount of label in thymine. This strain is phenotypically unstable producing cells resistant to bromodeoxyuridine (BrdU) and 6-thioguanine (6-TG) at high frequency. In contrast, the strain with the highest 5-methyldCMP deaminease, designated HAM⁺, is extremely stable, and the starting strain K1 HAM^s1 is intermediate between the HAM⁻ and HAM⁺ phenotypes. We have also shown that human diploid fibroblast strain MRC-5 has a phenotype like HAM⁺, whereas its SV40 transformed derivative, MRC-5V2 resembles HAM⁻ in having low 5-methyl dCMP deaminase activity, and is phenotypically unstable with regard to 6-TG resistance. It seems that 5-methyldCMP deaminase can be down-regulated in transformed cells, and this can promote de novo methylation by incorporation of 5-methyldCTP derived from 5-methyldCMP.     

Degradation of intracellular DNA in KB cells infected with cyt mutants of human adenovirus type 12.

A group of mutants (cyt mutants) with much reduced oncogenicity was isolated from the highly oncogenic human adenovirus type 12 (Takemori et al., Virology 36: 575-586, 1968). These mutants induce extensive cellular destruction during lytic infection of human cells and produce low yields of virions. We report here that human KB cells infected with cyt mutants synthesized a reduced amount of viral DNA as compared with cells infected with the parental virus. Furthermore, the newly synthesized viral and cellular DNAs were extensively degraded in mutant-infected cells. Viral DNA was first synthesized as complete genome size, and most of it was degraded to subgenomic size within 6 h after synthesis. This virus-induced DNA degradation function, as well as the low yield of virions, was prevented by co-infection with the parental virus.