Methylation of adenovirus genes in transformed cells and in vitro: influence on the regulation of gene expression?

An inverse correlation has been described between the levels of DNA methylation in specific segments of adenovirus DNA integrated into the genomes of transformed and tumor cells and the extent to which these segments are expressed as messenger RNA. In the adenovirus type 2 (Ad2)-transformed hamster cell lines HE2 and HE3, the virus-specific DNA binding protein (DBP) is not expressed, and the DNA in the DBP gene is completely methylated in all 5'-CCGG-3' sites. At least part of the late promoter/leader sequence of the DBP gene is present in cell lines HE2 and HE3. In line HE1, on the other hand, the DBP is expressed, and the DNA in the DBP gene is unmethylated at the 5'-CCGG-3' (HpaII) sites. The late promotor/leader sequence of the DBP gene is expressed in cytoplasmic RNA isolated from line HE1. The effect of DNA methylation has also been tested in vitro in a microinjection system using Xenopus laevis oocytes. Unmethylated DNA fragments of Ad2 (E2a region) have been found to serve as active templates. When the same fragments are methylated at the 5'-CCGG-3' sites by the HpaII DNA-methyltransferase, viral RNA synthesis is inhibited upon microinjection into oocyte nuclei. These results provide direct evidence for the notion that DNA methylated at highly specific sites is somehow involved in the regulation of gene expression.

Course of serum-Ig concentrations in B12 chickens of the UM line.

The University of Munich chicken line ("UM") is isogenic in respect to the MHC and divides into normogammaglobulinemic, permanent and transient dysgammaglobulinemic individuals. Hence the immune defect is independent of the MHC. Continual analysis of the immunoglobulins until the 50th week of life revealed: one group of dysgammaglobulinemic individuals showed an initial IgG peak between the third and sixth week of life. Unusually high IgM and IgA levels occur in permanent and transient dysgammaglobulinemic individuals previous to the appearance of the IgG deficit and previous to a possible initial IgG peak. These high levels remain throughout the life of the chicken, possibly due to a missing negative feedback mechanism. Transient dysgammaglobulinemic chickens also exhibited increased IgM and IgA values after IgG normalization. Based upon our results, we postulate that the dysgammaglobulinemia defect is already preprogrammed during late embryonic development. The prevalence of a B or T-cell defect is still under discussion.

The prophylactic use of antibiotics in cell culture.

This article describes the historical development of the prophylactic use of antibiotics in cell culture as well as their effects on cells. The influence of antibiotics on cell morphology, cellular degeneration and cell death and cellular function is summarized. Cellular DNA as well as protein synthesis are affected which can lead to interference with, or even changes in, metabolic processes. Such effects must be considered in cell culture research. As antibiotics are used in multifold ways, the otherwise standardized conditions in cell culture are no longer comparable. The prophylactic use of antibiotics is rejected for scientific reasons.

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.

Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells.

The establishment of de novo-generated patterns of DNA methylation is characterized by the gradual spreading of DNA methylation (I. Kuhlmann and W. Doerfler, J. Virol. 47:631-636, 1983; M. Toth, U. Lichtenberg, and W. Doerfler, Proc. Natl. Acad. Sci. USA 86:3728-3732, 1989; M. Toth, U. Müller, and W. Doerfler J. Mol. Biol. 214:673-683, 1990). We have used integrated adenovirus type 12 (Ad12) genomes in hamster tumor cells as a model system to study the mechanism of de novo DNA methylation. Ad12 induces tumors in neonate hamsters, and the viral DNA is integrated into the hamster genome, usually nearly intact and in an orientation that is colinear with that of the virion genome. The integrated Ad12 DNA in the tumor cells is weakly methylated at the 5'-CCGG-3' sequences. These sequences appear to be a reliable indicator for the state of methylation in mammalian DNA. Upon explantation of the tumor cells into culture medium, DNA methylation at 5'-CCGG-3' sequences gradually spreads across the integrated viral genomes with increasing passage numbers of cells in culture. Methylation is reproducibly initiated in the region between 30 and 75 map units on the integrated viral genome and progresses from there in either direction on the genome. Eventually, the genome is strongly methylated, except for the terminal 2 to 5% on either end, which remains hypomethylated. Similar observations have been made with tumor cell lines with different sites of Ad12 DNA integration. In contrast, the levels of DNA methylation do not seem to change after tumor cell explanation in several segments of hamster cell DNA of the unique or repetitive type. Restriction (HpaII) and Southern blot experiments were performed with selected cloned hamster cellular DNA probes. The data suggest that in the integrated foreign DNA, there exist nucleotide sequences or structures or chromatin arrangements that can be preferentially recognized by the system responsible for de novo DNA methylation in mammalian cells.

[Decubitus ulcer in paraplegic patients--a comparative clinico-pathologic study]. / Das Druckgeschwür bei Querschnittgelähmten--eine vergleichende klinisch-histologische Untersuchung.

331 pressure sores of 141 patients with paraplegia were investigated histologically, the results were partly correlated with the roentgenological features. The level of the spinal cord lesion was the segment Th12-L1 in 45%, the decubital ulcers were mostly localized above the os ischiadicum (64%). A pseudocancerosis was diagnosed in 14.2%, one time a cancer in a sacral decubitus has been found. The inflammation was limited to the soft tissue in 56.9%, in 28.5% a chronic osteomyelitis of different severity had developed. In 73.6% of the patients with a histologically proven osteomyelitis this osteomyelitis could be diagnosed roentgenologically too. The high amount and severity of the dermal and bony lesions justifies the radical surgical therapy of the pressure sores, which is practised today. Our investigations of chronic pressure sores did not point out new aspects of aetiology of pathogenesis--this is only possible by experimental models.

Tumor induction by human adenovirus type 12 in hamsters: loss of the viral genome from adenovirus type 12-induced tumor cells is compatible with tumor formation.

The patterns of integration of adenovirus type 12 (Ad12) DNA in 39 virus-induced hamster tumors were determined. Both the amount of Ad12 DNA persisting and the apparent sites of insertion differed from tumor to tumor. In 30 tumors, the intact Ad12 genome persisted in colinear arrangement and in multiple copies. In nine tumors, only the left- or the left- and right-hand parts of the Ad12 genome persisted in the tumor cells. In three other cell lines the Ad12 genomes were lost completely during continuous passage in culture. A shift from epithelioid to fibroblastic morphology correlated with loss of Adl2 genomes. The cell line H1111(1) derived from an Ad12-induced tumor had lost all viral DNA by the thirteenth subpassage, but was still oncogenic when reinjected into animals. This finding raises the question, to what extent persistence of the Ad12 genome is essential for the oncogenic phenotype. Tumor cells could be detected histologically inside local lymphatic vessels. In those experiments in which Ad12 preparations were used which contained sizeable proportions of the symmetric recombinant between Ad12 and KB cellular DNA (Deuring et al., 1981), tumors were observed in the nuchal region of the animals.

DNA methylation and viral gene expression in adenovirus-transformed and -infected cells.

The level of DNA methylation in adenovirus type 2 (Ad2) and type 12 (Ad12) DNA was determined by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI. As previously reported virion DNA of Ad2 and Ad12 is not methylated. Parental or newly synthesized Ad2 DNA in productively infected human KB or HEK cells is not methylated either, nor is the integrated form of Ad2 DNA in productively infected cells. Hamster cells and Muntiacus muntjak cells are abortively infected by Ad12. We have not detected methylation of Ad12 DNA in hamster or Muntiacus muntjak cells. An inverse correlation between the level of methylation and the extent of expression of viral DNA in Ad12-transformed hamster cells has been described earlier. A similar relation has been found for the EcoRI fragment B of Ad2 DNA which is not methylated but is expressed as the Ad2 DNA-binding (72K) protein in the Ad2-transformed hamster line HE1. Conversely, the same segment is completely methylated in lines HE2 and HE3, and there is apparently no evidence for the expression of the 72K protein in these cell lines.