BCL-xL: time-dependent dissociation between modulation of apoptosis and invasiveness in human malignant glioma cells.

Conditionally BCL-xL-overexpressing LNT-229 Tet-On glioma cell clones were generated to investigate whether the 'antiapoptosis phenotype' and the 'motility phenotype' mediated by BCL-2 family proteins in glioma cells could be separated. BCL-xL induction led to an immediate and concentration-dependent protection of LNT-229 cells from apoptosis. BCL-xL induction for up to 3 days did not result in altered invasiveness. In contrast, long-term BCL-xL induction for 21 days resulted in increased transforming growth factor-beta2 expression, and in metalloproteinase-2 and -14 dependent, but integrin independent, increased invasiveness. Withdrawal of doxycycline (Dox) abolished the protection from apoptosis whereas the 'invasion phenotype' remained stable. Dox stimulation of BCL-xL-inducible LNT-229 cells conferred infiltrative growth to BCL-xL-positive glioma cells in vivo and reduced the survival of tumor-bearing mice. These data allow to dissect a direct antiapoptotic action of BCL-xL from an indirect effect, presumably mediated by altered gene expression, which modifies tumor cell invasiveness in vitro and in vivo.

Tumorigenesis by adenovirus type 12 in newborn Syrian hamsters.

Ad12 oncogenesis in hamsters has been studied in detail to provide the following new data in this tumor model. Cells in the Ad12-induced tumors, often thought to be of neuronal origin, actually exhibit mesenchymal and neuronal characteristics and are probably of an undifferentiated derivation. Their intraperitoneal spread upon intramuscular injection of Ad12 adds another important new aspect. Differences in the integration patterns among the tumors suggest clonal origins from individual transformation events. Ad12 gene expression in the tumors is determined, at least in part, by the patterns of DNA methylation imprinted de novo upon the integrated Ad12 genomes. Differential Ad12 gene expression patterns, which have previously not been described in tumors, are an important parameter in Ad12 oncogenesis. The availability of cellular DNA arrays has opened up unprecedented possibilities to document changes in cellular transcription patterns, particularly of cancer-specific genes. These patterns exhibit differences and similarities among the different Ad12-induced tumors. Among the cellular genes, which are expressed in the Ad12-induced tumors, many are cancer-specific. We pursue the hypothesis that these alterations in cellular transcription patterns as a consequence of viral DNA integration and expression play an essential role in Ad12 oncogenesis.

Foreign DNA integration--perturbations of the genome--oncogenesis.

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 lambda, changes in cellular methylation and transcription patterns have been observed. Thus, the insertion of foreign DNA can have important functional consequences that are not limited to the site of foreign DNA insertion. These findings appear to be relevant also for tumor biology and for the interpretation of data derived from experiments with transgenic organisms. For most animals, the main portal of entry for foreign DNA is the gastrointestinal tract. Large amounts of foreign DNA are regularly ingested with the supply of nutrients. Starting in 1987/1988, we have been investigating the fate of orally administered foreign DNA in mice. Naked DNA of bacteriophage M13 and the cloned gene for the green fluorescent protein (GFP) of Aequorea victoria have been used as test molecules. Moreover, the plant-specific gene for the ribulose-1,5-bisphosphate carboxylase (rubisco) has been followed in mice after feeding soybean leaves. At least transiently, food-ingested DNA can be traced to different organs and, after transplacental transfer, to fetuses and newborns. There is no evidence for germ line transmission or for the expression of orally administered GFP DNA.

On the fate of plant or other foreign genes upon the uptake in food or after intramuscular injection in mice.

Uptake and persistence of the DNA of bacteriophage M13 and the cloned gene for the green fluorescent protein (GFP) as test genes for food-ingested DNA have previously been traced from the intestinal contents, via the gut wall, Peyer's patches and peripheral white blood cells to spleen and liver, and via the placenta to fetuses and newborn animals. We have now chosen a natural scenario and fed soybean leaves to mice. The distribution of the plant-specific, nucleus-encoded ribulose-1,5-bisphosphate carboxylase (Rubisco) gene has been studied in the mouse. The Rubisco gene or fragments of it can be recovered in the intestine from 2 h up to 49 h after feeding, and in the cecum up to 121 h after ingestion. Thus, plant-associated, naturally fed DNA is more stable in the intestinal tract than naked DNA. Rubisco gene-specific PCR products have also been amplified from spleen and liver DNA. There is no evidence for the expression of orally administered genes, as assessed by the RT-PCR method. Moreover, mice have been continuously fed daily with GFP DNA for 8 generations and have been examined for the transgenic state by assaying DNA isolated from tail tips, occasionally from internal organs of the animals, by PCR. The results have been uniformly negative and argue against the germline transfer of orally administered DNA. Upon the intramuscular injection of GFP DNA, authentic GFP DNA fragments have been amplified by PCR from DNA from muscle for up to 17 months post-injection, and from DNA from organs remote from the site of injection up to 24 h post injection. GFP fragments can also be retrieved from the intestinal contents up to 6 h post injection. The organism apparently eliminates injected foreign DNA via the liver-bile-intestinal route.

The fate of foreign DNA in mammalian cells and organisms.

We have investigated the consequences of foreign DNA insertions into the genomes of mammalian cells in transgenic cell lines, in adenovirus type 12 (Ad12)-transformed cells, in Ad12-induced tumor cells or in transgenic mice. We have reported previously on the de novo methylation of integrated foreign genomes and on extensive changes in cellular patterns of DNA methylation upon foreign DNA insertion. These studies have been extended and several independent methods have been applied to document these alterations in cellular DNA methylation and gene expression patterns in transgenic cell lines and in transgenic mice. These data are relevant for the mechanism of (viral) oncogenesis and for the interpretation of data gathered in experiments with transgenic animals.

On the fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus.

We have previously shown that, when administered orally to mice, bacteriophage M13 DNA, as a paradigm foreign DNA without homology to the mouse genome, can persist in fragmented form in the gastrointestinal tract, penetrate the intestinal wall, and reach the nuclei of leukocytes, spleen and liver cells. Similar results were obtained when a plasmid containing the gene for the green fluorescent protein (pEGFP-C1) was fed to mice. In spleen, the foreign DNA was detected in covalent linkage to DNA with a high degree of homology to mouse genes, perhaps pseudogenes, or to authentic E. coli DNA. We have now extended these studies to the offspring of mice that were fed regularly during pregnancy with a daily dose of 50 microg of M13 or pEGFP-C1 DNA. Using the polymerase chain reaction (PCR) or the fluorescent in situ hybridization (FISH) method, foreign DNA, orally ingested by pregnant mice, can be discovered in various organs of fetuses and of newborn animals. The M13 DNA fragments have a length of about 830 bp. In various organs of the mouse fetus, clusters of cells contain foreign DNA as revealed by FISH. The foreign DNA is invariably located in the nuclei. We have never found all cells of the fetus to be transgenic for the foreign DNA. This distribution pattern argues for a transplacental pathway rather than for germline transmission which might be expected only after long-time feeding regimens. In rare cells of three different fetuses, whose mothers have been fed with M 13 DNA during gestation, the foreign DNA was detected by FISH in association with both chromatids. Is maternally ingested foreign DNA a potential mutagen for the developing fetus?