DNA released from neutrophil extracellular traps (NETs) activates pancreatic stellate cells and enhances pancreatic tumor growth.

Neutrophil extracellular trap (NET) formation results in the expulsion of granulocyte proteins and DNA into the extracellular space. This process is mediated by the enzyme peptidyl arginine deiminase 4 (PADI4) and translocation of elastase to the nucleus. NET formation, marked by increased levels of extracellular DNA, promotes pancreatic cancer proliferation and metastasis. Mice deficient in Padi4 demonstrate decreased pancreatic tumor growth, associated with a reduction in circulating extracellular DNA levels, diminished pancreatic stromal activation and improved survival in murine orthotopic pancreatic adenocarcinoma. Transplantation of Padi4-/- bone marrow into genetically engineered mice with Kras driven pancreatic adenocarcinoma (Pdx1-Cre:KrasG12D/+, KC mice) limits the frequency of invasive cancers when compared with syngeneic controls. DNA from neutrophils activates pancreatic stellate cells that form dense, fibrous stroma which can promote and enable tumor proliferation. DNase treatment diminishes murine tumor growth and stromal activation to reverse the effect of NETs within the tumor microenvironment. Furthermore, deletion of the receptor for advanced glycation end products (RAGE) in pancreatic stellate cells abrogates the effects of DNA in promoting stellate cell proliferation and decreases tumor growth. Circulating neutrophil-derived DNA correlates with the stage in patients with pancreatic ductal adenocarcinoma, confirming the role of NETs in human pancreatic cancer. These findings support further investigation into targeting of NETs, PADI4 and extracellular DNA as a potential treatment strategy in patients with pancreatic cancer. Trial Registration: This study reports correlative data from a clinical trial registered with clinicaltrials.gov, NCT01978184 (November 7, 2013).

Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps.

BACKGROUND: The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis. METHODS: Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated. RESULTS: The addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE. CONCLUSION: NETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer. TRIAL REGISTRATION: This study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013).

Nucleotide sequence comparisons between several strains and isolates of human cytomegalovirus reveal alternate start codon usage.

Mutations abound in all viral populations, which are thus rendered adaptable to changes in environmental conditions. Human cytomegalovirus (HCMV) is an important human pathogen for investigating nucleotide sequence variations because they can affect its potential to cause disease. We have determined part of the nucleotide sequence of the Toledo strain and compared it to the published sequences of the strains AD169, Toledo, and Towne and of three clinical isolates. Overall nucleotide sequence divergence between strains AD169 and Toledo amounts to roughly 2%, with considerable variations across the viral genome. In aligning the Toledo nucleotide sequences with those of the other strains and clinical isolates, numerous amino-terminal extensions of the known open reading frames (ORFs) have been noted. These extensions carry additional AUG or non-canonical CUG or GUG translational initiation codons. CUG and GUG have previously been shown to serve as translational start codons in prokaryotic and eukaryotic systems. Six of the more closely inspected extensions start with an AUG, 26 with a CUG, and 26 with a GUG. Some of these extended sequences might bestow altered biological properties upon HCMV proteins. These ORF extensions are common to the sequenced genomes of most of the HCMV strains or isolates. Supporting evidence for their functionality comes from studies on HCMV mRNAs that were isolated from HCMV-infected human cells. Several of these viral mRNA sequences carry the identified ORF extensions. Moreover, in the amino-terminal ORF extensions, codon usage in general resembles that in the main parts of several of the HCMV genes analyzed for this property.

De novo methylation, long-term promoter silencing, methylation patterns in the human genome, and consequences of foreign DNA insertion.

This chapter presents a personal account of the work on DNA methylation in viral and mammalian systems performed in the author's laboratory in the course of the past 30 years. The text does not attempt to give a complete and meticulous account of the work accomplished in many other laboratories; in that sense it is not a review of the field in a conventional sense. Since the author is also one of the editors of this series of Current Topics in Immunology and Microbiology on DNA methylation, to which contributions by many of our colleagues in this field have been invited, the author's conscience is alleviated that he has not cited many of the relevant and excellent reports by others. The choice of viral model systems in molecular biology is well founded. Over many decades, viruses have proved their invaluable and pioneering role as tools in molecular genetics. When our interest turned to the demonstration of genome-wide patterns of DNA methylation, we focused mainly on the human genome. The following topics in DNA methylation will be treated in detail: (1) The de novo methylation of integrated foreign genomes; (2) the long-term gene silencing effect of sequence-specific promoter methylation and its reversal; (3) the properties and specificity of patterns of DNA methylation in the human genome and their possible relations to pathogenesis; (4) the long-range global effects on cellular DNA methylation and transcriptional profiles as a consequence of foreign DNA insertion into an established genome; (5) the patterns of DNA methylation can be considered part of a cellular defense mechanism against foreign or repetitive DNA; which role has food-ingested DNA played in the elaboration of this mechanism? The interest in problems related to DNA methylation has spread-like the mechanism itself-into many neighboring fields. The nature of the transcriptional programs orchestrating embryonal and fetal development, chromatin structure, genetic imprinting, genetic disease, X chromosome inactivation, and tumor biology are but a few of the areas of research that have incorporated studies on the importance of the hitherto somewhat neglected fifth nucleotide in many genomes. Even the fly researchers now have to cope with the presence of this nucleotide, in however small quantities it exists in the genome of their model organism, at least during embryonal development. The bulk of the experimental work accomplished in the author's laboratory has been shouldered by many very motivated undergraduate and graduate students and by a number of talented postdoctoral researchers. Their contributions are reflected in the list of references in this chapter. We have also had the good luck to receive funding through a number or organizations as acknowledged.

On the biological significance of DNA methylation.

This chapter presents a personal account of the work on DNA methylation in viral and mammalian systems performed in the author's laboratory in the course of the past thirty years. The text does not attempt to give a complete and meticulous account of the many relevant and excellent reports published by many other laboratories, so it is not a review of the field in a conventional sense. The choice of viral model systems in molecular biology is well founded. Over many decades, viruses have proven their invaluable and pioneering role as tools in molecular genetics. When our interest turned to the demonstration of genome-wide patterns of DNA methylation, we focused mainly on the human genome. The following topics in DNA methylation will be treated in detail: (i) the de novo methylation of integrated foreign genomes; (ii) the long-term gene silencing effect of sequence-specific promoter methylation and its reversal; (iii) the properties and specificity of patterns of DNA methylation in the human genome and their possible relations to pathogenesis; (iv) the long-range global effects on cellular DNA methylation and transcriptional profiles as a consequence of foreign DNA insertion into an established genome; (v) the patterns of DNA methylation can be considered part of a cellular defense mechanism against foreign or repetitive DNA; what role has food-ingested DNA played in the elaboration of this mechanism?

Localization of integrated adenovirus DNA in the hamster genome.

Adenovirus DNA integrated into the genomes of adenovirus-transformed hamster cells or of adenovirus type 12 (Ad12)-induced hamster tumor cells can be located at many different chromosomal sites. This raises the question as to whether distinct isochores of the hamster cell genome might be more accessible to recombination with adenovirus DNA. In Ad12- or Ad2-transformed hamster cell lines, and in Ad12 revertants, the investigated integrated viral DNA sequences were assigned to isochore families by analyzing DNA fractions from preparative CsCl density gradients for their buoyant densities (and, therefore, GC levels) and for the presence of viral DNA. Adenovirus DNA sequences were found in different isochores, which did not generally match the base composition of viral sequences. This is in apparent contrast to what was previously observed with retroviral integration. However, in cell lines carried in culture for many years, the viral DNA sequences might have been transposed to different isochore positions, since the host sequences flanking the viral DNA appear to have been conserved.

Quantitative c-erbB-2 but not c-erbB-1 mRNA expression is a promising marker to predict minor histopathologic response to neoadjuvant radiochemotherapy in oesophageal cancer.

We examined the potential of quantitative epidermal growth factor receptor (EGFR, synonym: c-erbB-1) and c-erbB-2 (synonym: HER2/neu) mRNA expression to predict minor or major histopathologic response to neoadjuvant radiochemotherapy (cis-platinum, 5-FU, 36 Gy), followed by radical surgical resection, in patients with oesophageal cancer. Tissue samples were collected by endoscopic biopsy prior to treatment. RNA was isolated from biopsies and quantitative real-time reverse transcriptase-polymerase chain reaction assays were performed to determine c-erbB-1 and c-erbB-2 mRNA expression. Relative expression (tumour/paired normal tissue ratio standardised for beta-actin) was calculated for EGFR and c-erbB-2 mRNA. Expression levels were correlated with the objective histopathologic response in resected specimens. Histomorphologic regression was defined as major response when resected specimens contained less than 10% of residual vital tumour cells, or in case a pathologically complete response was achieved. Expression of c-erbB-1 mRNA was not associated with the degree of histomorphological response. In contrast, the relative expression levels of c-erbB-2 mRNA >1 were not associated with major histopathologic responses (sensitivity 41.6%, specificity 100%), and 10 out of 36 (28%) patients could be unequivocally identified, whose tumours did not respond well to the delivered neoadjuvant radiochemotherapy (P<0.01). Quantitative expression levels of c-erbB-2, but not c-erbB-1 mRNA, in pretreatment biopsies appear to predict minor histopathologic response to our neoadjuvant radiochemotherapy protocol. This test could be used to prevent expensive, non effective and potentially harmful therapies in approximately one-fourth of our patients, and leads to a more individualised type of combined modality treatment.

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.

The gastrointestinal tract as the portal of entry for foreign macromolecules: fate of DNA and proteins.

The gastrointestinal tract (GIT) of mammals is the main portal of entry for foreign DNA and proteins. We have documented the fate of orally administered DNA or protein in the GIT of the mouse. The gene for the Green Fluorescent Protein (GFP) (4.7 kb) and the genomes of bacteriophage M13 (7.25 kb) and adenovirus type 2 (Ad2; 35.9 kb) were used as test DNAs. Persistence of these DNAs in the GIT was monitored by Southern hybridization and fluorescent in situ hybridization (FISH) or by PCR. For studies on proteins, recombinant glutathione-S-transferase was fed to mice. Survival of the protein in the GIT was then assessed by Western blotting. Depending on feeding schedules and food regimens, but irrespective of mouse strain or DNA length, fragments of the GFP gene or other DNAs were detectable for up to 18 h after feeding by Southern blot analysis. The GFP DNA could be visualized by FISH in cecal epithelia. A high fiber diet reduced the time required for food to pass through the GIT, and foreign DNA was cleared more rapidly. A high fat diet or complexing of the foreign DNA with protamine or lipofectin did not extend DNA persistence times. Undegraded GST protein was detected only in foregut contents up to 30 min after feeding. At 15 and 30 min post feeding, trace amounts of GST were found in extracts of the kidney. The GIT is constantly exposed to highly recombinogenic fragments of foreign DNA and to intact foreign proteins. Our data have implications for studies on carcinogenesis and mutagenesis, and on the pathogenicity of infectious proteins such as prions.

The abortive infection of Syrian hamster cells with human adenovirus type 12.

Human adenovirus type 12 (Ad12) induces undifferentiated tumors in newborn Syrian hamsters, and this tumor model has been investigated in detail in our laboratory. One of the characteristics of the Ad12-hamster cell system is a strictly abortive infection cycle. In this chapter, we summarize previous and more recent results of studies on the interaction of Ad12 with the nonpermissive BHK21 hamster cell line. The block of Ad12 replication lies before viral DNA replication and late gene transcription which cannot be detected with the most sensitive techniques. Ad12 adsorption, cellular uptake and transport of the viral DNA to the nucleus are less efficient in the nonpermissive hamster cells than in permissive human cells. However, most of the early functions of the Ad12 genome are expressed in BHK21 cells, though at a low level. In the downstream region, the first exon, of the major late promoter (MLP) of Ad12 DNA, a mitigator element of 33 nucleotide pairs in length has been identified which contributes to the inactivity of the MLP in hamster cells and its markedly decreased activity in human cells. The E1 functions of Ad2 or Ad5 are capable of partly complementing the Ad12 deficiencies in hamster cells in that Ad12 viral DNA replication and late gene transcription can proceed, e.g. in a BHK hamster cell line, BHK297-C131,which carries in an integrated form and constitutively expresses the E1 region of Ad5 DNA. Nevertheless, the late Ad12 mRNAs, which are synthesized in this system with the authentic nucleotide sequence, fail to be translated to structural viral proteins. Hence, infectious virions are not produced in the partly complementing system. Probably there is also a translational block for late Ad12 mRNAs in hamster cells. We have recently shown that the overexpression of the Ad12 preterminal protein (pTP) gene or of the E1A gene facilitates the synthesis of full-length, authentic Ad12 DNA in BHK21 cells infected with Ad12. Apparently the pTP has a hitherto unknown function in eliciting full cycles of Ad12 DNA replication even in nonpermissive BHK21 cells when sufficient levels of Ad12 pTP are produced. We pursue the possibility that the completely abortive infection cycle of Ad12 in hamster cells ensures the survival of Ad12-induced hamster tumor cells which all carry, integrated in their genomes, multiple copies of Ad12 DNA. In this way, the viral genomes are immortalized and expanded in a huge number of tumor cells.

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.

Cellular and early viral factors in the interaction of adenovirus type 12 with hamster cells: the abortive response.

The interaction of human adenovirus type 12 (Ad12) with Syrian hamster cells is remarkable in that there is a block of viral DNA replication and late gene transcription. We have screened several cellular factors known to play a role in adenovirus replication for their possible contributions to the interactions of Ad12 in the abortive BHK21 hamster cell system. (1) Western blot analyses of total protein extracts from Ad12- or Ad2-infected BHK21 cells do not reveal a significant difference in the accumulation of NFIII protein at different times after infection. Transcriptional levels of the NFIII gene in BHK21 cells are not altered upon the abortive infection with Ad12 or the productive infection with Ad2. The amount of NFIII protein is markedly reduced in nuclear extracts from BHK21 cells as compared with extracts from C131 hamster cells or human HeLa cells. A presumptive defect in NFIII transport to the nuclei rather than overall reduced NFIII gene transcription might explain the low abundance of NFIII in the nuclei of uninfected or Ad12-infected BHK21 cells. The productive infection of BHK21 or C131 cells with Ad2 leads to an increase in the NFIII concentration in the nuclei of infected cells, late after infection to a decrease; (2) NFI levels in the nuclei of mock-infected or Ad2- or Ad12-infected BHK21 cells are comparable with those in HeLa or in C131 cells. Thus, deficiencies in NFI may not play a role in the abortive system; (3) The absence of morphological alterations in PML protein domains from globular to track-like structures in the nuclei of Ad12-infected hamster cells correlates with the inability of Ad12 DNA to replicate in BHK21 cells. In BHK21 cells, the E4-ORF3 of Ad12 DNA is only weakly transcribed and only small amounts of the gene product are synthesized. In Ad12-infected C131 cells, which allow the replication of Ad12 DNA, the E4-ORF3 of Ad12 DNA is expressed, and track-like PML protein structures are observed. Transfection of the 12-E4-ORF3-EGFP construct leads to the expression of both the green fluorescent protein (GFP) and of the 12-E4-ORF3 gene product in 20-30% of the transfected BHK21 cells and elicits the morphological reorganization of the PML protein structures in the successfully transfected BHK21 cells. Similar results are obtained upon transfection of the 2-E4-ORF3 construct. Untransfected cells or cells transfected with the empty pIRES2-pEGFP vector carry the globular PML protein phenotype; (4) The expression of the 12-E4-ORF3-EGFP and/or of the NFIII-EGFP constructs upon transfection following Ad12-infection of BHK21 cells fails to promote Ad12 DNA replication. Hence, the formation of track-like PML protein structures in BHK21 cells by itself is not a sufficient precondition for Ad12 DNA replication in this abortive system. The data demonstrate that the expression of NFI, NFIII, and/or the conversion of the PML domains do not suffice to elicit Ad12 DNA replication in the abortive hamster cell system.

Overexpression of the adenovirus type 12 (Ad12) pTP or E1A gene facilitates Ad12 DNA replication in nonpermissive BHK21 hamster cells.

In the adenovirus type 12 (Ad12) hamster cell system, abortive virus infection is one of the factors associated with the highly efficient oncogenesis in newborn Syrian hamsters. We have shown earlier that the replication and efficient late transcription of the Ad12 genome are blocked in Syrian hamster cells. Some of the early Ad12 functions are transcribed in these cells, although at a minimal rate. In the present study, we demonstrate that low expression levels of the E1A and precursor to terminal protein (pTP) genes of Ad12 seem to be responsible for the lack of Ad12 DNA replication in hamster cells. The Ad12 genes for the E1A functions or for pTP were tethered to the strong early promoter of the human cytomegalovirus and transfected into BHK21 cells. Subsequently, these cells were infected with Ad12 virions. In Ad12-infected BHK21 cells, which overexpressed pTP or E1A, full-length Ad12 DNA was de novo synthesized, as documented by metabolic labeling with [3H]thymidine and by zone velocity sedimentation in alkaline sucrose gradients followed by gel electrophoresis of the 3H-labeled DNA and Southern blot hybridization to 32P-labeled Ad12 DNA. Transfection of the cloned E1A region of Ad2 yielded similar results. The newly synthesized Ad12 DNA was covalently linked to pTP. The Ad12 DNA binding protein (DBP) and DNA polymerase (pol) genes were transcribed at levels similar to those in merely Ad12-infected cells. In pTP or E1A gene-transfected and Ad12-infected BHK21 cells, marginal levels of late Ad12 mRNA were detectable. Late Ad12 proteins were, however, not synthesized. Apparently, Ad12 DNA replication in hamster cells is rendered impossible due to insufficient threshold levels of the viral E1A and/or pTP.

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.

Foreign DNA integration. Genome-wide perturbations of methylation and transcription in the recipient genomes.

In hamster cells transgenic for the DNA of adenovirus type 12 (Ad12) or for the DNA of bacteriophage lambda, the patterns of DNA methylation in specific cellular genes or DNA segments remote from the site of transgene insertion were altered. In the present report, a wide scope of cellular DNA segments and genes was analyzed. The technique of methylation-sensitive representational difference analysis (MS-RDA) was based on a subtractive hybridization protocol after selecting against DNA segments that were heavily methylated and hence rarely cleaved by the methylation-sensitive endonuclease HpaII. The MS-RDA protocol led to the isolation of several cellular DNA segments that were indeed more heavily methylated in lambda DNA-transgenic hamster cell lines. By applying the suppressive subtractive hybridization technique to cDNA preparations from nontransgenic and Ad12-transformed or lambda DNA-transgenic hamster cells, several cellular genes with altered transcription patterns were cloned from Ad12-transformed or lambda DNA-transgenic hamster cells. Many of the DNA segments with altered methylation, which were isolated by a newly developed methylation-sensitive amplicon subtraction protocol, and cDNA fragments derived from genes with altered transcription patterns were identified by their nucleotide sequences. In control experiments, no differences in gene expression or DNA methylation patterns were detectable among individual nontransgenic BHK21 cell clones. In one mouse line transgenic for the DNA of bacteriophage lambda, hypermethylation was observed in the imprinted Igf2r gene in DNA from heart muscle. Two mouse lines transgenic for an adenovirus promoter-indicator gene construct showed hypomethylation in the interleukin 10 and Igf2r loci. We conclude that the insertion of foreign DNA into an established mammalian genome can lead to alterations in cellular DNA methylation and transcription patterns. It is conceivable that the genes and DNA segments affected by these alterations depend on the site(s) of foreign DNA insertion.

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.

Cognitive and behavioral profile of fragile X boys: correlations to molecular data.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation after Down syndrome. The expansion of a CGG repeat, located in the 5'-untranslated region (5'-UTR) of the FMR1 (fragile X mental retardation) gene, leads to the hypermethylation of the repeat and the upstream CpG island. Methylation is associated with transcriptional silencing of the FMR1 gene. The lack of FMR1 protein is believed to be responsible for the typical physical and mental characteristics of the syndrome. To analyze the specific phenotype of that syndrome as well as possible associations between the phenotype and the genotype, we examined a group of 49 fragile X boys and a control group of 16 patients with tuberous sclerosis. To determine the cognitive and behavioral phenotype, the Kaufman Assessment Battery for Children (K-ABC), the Child Behavior Checklist (4/18), and a structured psychiatric interview (Kinder DIPS) were used. The genotype was analyzed by the Southern blot method. The phenotype of boys with FXS is characterized by a specific cognitive profile with strengths in acquired knowledge and in simultaneous processing. The psychiatric comorbidity is high and ADHD (attention deficit hyperactivity disorder), oppositional defiant disorder, enuresis, and encopresis predominate. In a group of 24 fragile X boys, no significant correlations between the specific aspects of the phenotype and the genotype were found.

Familial form of hirschsprung disease: nucleotide sequence studies reveal point mutations in the RET proto-oncogene in two of six families but not in other candidate genes.

Hirschsprung disease (HSCR; McKusick 142623) or aganglionic megacolon is a frequent (1 in 5,000 live births) heritable disorder of the enteric nervous system. By haplotyping with a variety of microsatellite markers, by amplifying all 20 exons of the RET proto-oncogene and by applying a direct DNA sequencing protocol, we have analyzed the DNA from HSCR patients in 6 different families. In one family with a joint occurrence of HSCR and FMTC (follicular medullary thyroid carcinoma), we have identified a mutation in codon 609 in one out of 6 cysteine residues encoded in exon 10 of the RET gene. This C609R point mutation has not previously been reported to cause HSCR. In 2 of the HSCR patients described here from different families, we have found a mutation in exon 2 (R77C) and a silent mutation in exon 3 (Y204Y), respectively, in the extracellular part of the RET proto-oncogene. In introns 2 and 17 of the RET proto-oncogene in 2 families, we have detected single nucleotide exchanges that are probably polymorphisms with unknown, if any, relations to HSCR. The DNA sequences of 5 further genes (GDNF, GDNFRalpha, EDN3, EDNRB, and NTN), that may contribute to the development of HSCR, have not shown mutations in the patients analyzed so far. In 2 of the reported families with several affected children and one grandchild, sequence analyses revealed no mutations in the coding regions of any of the candidate genes analyzed.