Nuclear methylation levels in normal and cancerous thyroid cells.

Background: Most cancers show abnormal DNA methylation and a positive correlation between hypomethylation and tumour progression.

Patients And Methods: In our laboratory the extent of DNA methylation in individual nuclei in normal, cancer and non-cancer thyroid tissue samples was quantified according to a previously described method of computer-assisted semi-quantitative analysis. Cancer and non-cancer samples were obtained from nine patients with different thyroid pathologies (one multinodular goitre and eight carcinomas).

DNA demethylation is directly related to tumour progression: evidence in normal, pre-malignant and malignant cells from uterine cervix samples.

A computer-assisted assay based on the quantitative analysis of DNA methylation in individual interphase nuclei by indirect immunolabelling with anti-5-methylcytosine antibodies was recently developed in our laboratory. In situ analyses were performed on individual nuclei from normal and experimentally hypo- or hypermethylated cultured cells as well as on human peripheral blood B-lymphocytes from normal and chronic lymphoid leukemia (CLL) samples. We present the results obtained on cells from patients affected by different degrees of preneoplastic or neoplastic changes of the uterine cervix as compared to normal controls.

Evaluation of global DNA hypomethylation in human colon cancer tissues by immunohistochemistry and image analysis.

Background: Global hypomethylation of DNA is frequently observed in human tumours. This alteration is detected in early adenomas in colorectal tumorigenesis. Information is currently acquired after extraction of DNA from tissues, digestion with nucleases, and analysis by reverse phase chromatography, or treatment with restriction enzymes followed by gel electrophoresis analysis and Southern hybridisation with radiolabelled probes.

DNA global hypomethylation in EBV-transformed interphase nuclei.

In tumors, DNA is often globally hypomethylated compared to DNA extracted from normal tissues. This observation is usually made after extraction and exhaustive digestion of DNA followed by analysis of nucleosides by chromatography or digestion with restriction enzymes, gel analysis, and hybridization. This approach provides an average value which does not give information on the various cell subpopulations included in heterogeneous samples.

Reduced levels of poly(ADP-ribosyl)ation result in chromatin compaction and hypermethylation as shown by cell-by-cell computer-assisted quantitative analysis.

The unmethylated status of the CpG islands is important for gene expression of correlated housekeeping genes since it is well known that their methylation inhibits transcription process. An interesting question that has been discussed but not solved is how the CpG islands maintain their characteristic unmethylated status even though they are rich in CpG dinucleotides. Our previous in vitro and in vivo research has shown that poly(ADP-ribosyl)ation is involved in protecting CpG dinucleotides from full methylation in genomic DNA and that a block of poly(ADP-ribosyl)ation is also involved in modifying the methylation pattern in the promoter region of Htf9 housekeeping gene.

Computer-assisted analysis of methylation status of individual interphase nuclei in human cultured cells.

This paper demonstrates that (a) differences in the methylation levels of interphase nuclei can be measured on a cell-by-cell basis, (b) the binding sites of beta-satellite DNA and 5-methylcytosine (5MeC)-rich regions can be localised in interphase nuclei and metaphase chromosomes by sequential in situ hybridization and indirect immunolabelling, and (c) quantitative differences in the relative extensions of beta-satellite DNA and anti-5MeC antibody binding areas can also be measured. This goal was achieved by indirect immunolabelling by anti-5MeC antibodies (Reynaud et al.: Cancer Lett.

Unusual segregation of t(11;22) resulting from crossing-over followed by 3:1 disjunction at meiosis I.

Reciprocal translocation t(11;22)(q23;q11) is of particular interest because the unbalanced offspring of the translocation carriers usually present with a supernumerary derivative chromosome 22. This common unbalanced karyotype is the result of 3:1 chromosome segregation during meiosis. We report the third case of a rare segregation pattern of a paternal 11;22 translocation.

Cytological evidence for 5-azacytidine-induced demethylation of the heterochromatic regions of human chromosomes.

This work was aimed at studying the effects of the demethylating agent 5-azacytidine (5-azaC) on the constitutive heterochromatin of human chromosomes at the cytological level. Metaphase preparations from peripheral blood lymphocyte and lymphoblastoid cultures obtained by standard methods were treated with the agent. Labelling of the heterochromatic regions was achieved by the indirect immunostaining method using anti-5-methylcytosine (5MeC) monoclonal antibodies and peroxidase-tagged second antibodies.

Persistence of increased levels of ribosomal gene activity in CHO-K1 cells treated in vitro with demethylating agents.

The rate of ribosomal gene activity was evaluated by silver staining of the Nucleolus Organisers (NOs) in cultured CHO-K1 cells after a 12 h pulse with two demethylating agents (L-ethionine and 5-azacytidine). Silver staining of the NOs was measured every 24 h, from 24 up to 110 h after seeding. The purpose was to test the hypothesis that drug-induced demethylation is associated to heritable modifications of rDNA activity.

Chromosome length and DNA loop size during early embryonic development of Xenopus laevis.

The looped organization of the eukaryotic genome mediated by a skeletal framework of non-histone proteins is conserved throughout the cell cycle. The radial loop/scaffold model envisages that the higher order architecture of metaphase chromosomes relies on an axial structure around which looped DNA domains are radially arranged through stable attachment sites. In this light we investigated the relationship between the looped organization and overall morphology of chromosomes.

Inheritance of ribosomal gene activity and level of DNA methylation of individual gene clusters in a three generation family.

Ribosomal gene activity and levels of DNA methylation were investigated by cytochemical and immunological methods in the nucleolar organizer regions (NORs) of individually recognised acrocentric chromosomes. Mendelian inheritance of ribosomal gene activity in a three generation family was demonstrated, together with consistent behaviour of individual gene clusters in different carriers, even when environmental conditions were changed. For most chromosomes, an inverse relationship between gene activity and the level of DNA methylation was observed.

Relationship between the number and function of human ribosomal genes.

The relative number of ribosomal RNA genes of the acrocentric chromosomes in one individual was measured by counting grains after in situ hybridization of 3H-labeled human 18S rDNA to fixed metaphase chromosomes. The relative amount of ribosomal RNA gene activity of each of the same chromosomes was estimated by determining the frequency with which the chromosome's nucleolus organizer region (NOR) was silver stained, the size of the silver-stained region, and how often the chromosome was found in satellite association. Results were similar in phytohemagglutinin-stimulated T-lymphocytes, Epstein-Barr virus transformed lymphoblasts, and fibroblasts.

Laron dwarfism: cellular unresponsiveness to GH demonstrated on cultured lymphocytes by a cytochemical method.

The response to growth hormone (GH) of cultured lymphocytes from three patients with Laron dwarfism (LD), one subject with growth hormone deficiency and a normal adult volunteer was examined by employing the cytochemical method of selective silver staining that evidentiates the chromosomal sites of those gene clusters (Nucleolus Organizers, NOs) which are actively involved in rRNA transcription. Lymphocytes from the normal donor responded to GH administration with a significant increase of the mean number of silver positive NOs per cell as well as lymphocytes from the growth hormone deficient patient (P less than 0.001).

Differential ribosomal gene responsiveness to human growth hormone is visualized by selective silver staining.

Differential activity of rRNA gene clusters following growth-hormone administration has been demonstrated in cultured lymphocytes from subjects with different genetic backgrounds, i.e., with or without in vivo peripheral responsiveness to the hormone.

Hormone-modulated rRNA gene activity is visualized by selective staining of the NOs.

The role of Growth Hormone and Dexamethasone in the regulation of rRNA gene activity was evaluated on cultured human fibroblasts by the cyto-chemical method of selective silver staining. By this method the transcriptionally active r-gene clusters can be specifically visualized in individual cells. Statistically significant increases in the rate of rRNA transcriptional activity were demonstrated after hormone administration.

Cytologic evidence for increased rRNA gene activity in hemin-induced K562(S) cells.

Hemin-induced K562(S) cells have been studied for the following parameters: cell proliferation, erythroid induction, hemoglobin accumulation, and activation of ribosomal gene clusters 48 hr after hemin induction. Increased transcriptional activity of rRNA genes has been demonstrated by cytochemical methods at both the cell population and single cell level. The following results have been obtained: (a) The vast majority of induced cells shows a highly significant increase in the number of active rRNA gene clusters per cell.

The transcriptional activity of individual ribosomal DNA gene clusters is modulated by serum concentration.

The activity of ribosomal gene clusters has been studied by cytological methods in human cultured cells grown in different amounts of serum under controlled experimental conditions. It has been shown that increasing amounts of serum induce an increase in ribosomal RNA synthesis at the single cell level. Furthermore, the concomitant identification of individual rRNA gene clusters by fluorescent techniques allowed us to demonstrate: (1) that individual gene clusters have differential transcriptional activity and differential frequency of activation; (2) that ribosomal gene activity is closely associated with the amount of silver-positive gene product and; (3) that environmental variations modulate rRNA synthesis by repressing or derepressing specific gene clusters.

Cytologic demonstration of differential activity of rRNA gene clusters in different human tissues.

rRNA gene activity was evaluated by cytologic methods in cultured human cells from two different tissues grown under controlled experimental conditions. The modal and average numbers of silver positive nucleolus organizers (NOs) per cell as well as the distribution of cells with different numbers of silver positive NOs and different combinations of D- plus G-group silver stained chromosomes, were evaluated. Statistically significant differences in the average number of silver positive NOs per cell between leukocytes and fibroblasts grown under standard experimental conditions have been demonstrated.

Analysis of the DNA replication pattern of a translocation (tX/X, qter----p221::p223----qter) chromosome in leukocyte and fibroblast cultures.

The results of a detailed analysis of DNA replication in a late replicating tX/X chromosome (qter----p221::p223----qter) are reported. The chronology of DNA replication has been analyzed by comparing (a) the replication patterns of each of the two moieties of the translocation chromosome in different cells and (b) the two moieties with each other in the same cell. The study has been done on leukocyte and fibroblast cultures after BUdR incorporation.

Silver positivity of the NORs during embryonic development of Xenopus laevis.

Transcriptional activity of ribosomal RNA (rRNA) genes is detectable around blastula-gastrula transition during the embryonic development of amphibians and other non-mammalian systems. The silver staining reaction, known to selectively stain transcriptionally active nucleolus organizer regions (NORs) both in interphase and metaphase chromosomes allowed us to follow the activation of the NORs during the embryonic development of Xenopus laevis.

Screening for cytogenetic polymorphisms in a random sample of liveborn infants from Italian population.

The frequency of major and minor chromosome variants is studied in a random sample of newborns in Central Italy. Special attention is paid to the objective criteria used to evaluate minor variants. In our sample, the frequency of acrocentric chromosome variants is found to be unusually high compared with previous studies.