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. AIMS: The purpose of our work was to evaluate the global methylation status of DNA in malignant lesions without loosing the histopathological features of the samples. PATIENTS: The investigation was performed on paired normal-tumour tissues from 13 patients undergoing surgical resection of colorectal adenocarcinomas. METHODS: Antibodies raised against 5-methylcytidine can be used to label methyl rich regions in interphase nuclei. This technique was adapted to the study of paraffin embedded tissues and an immunohistochemical method was developed to assess the global methylation status of individual nuclei while preserving cell morphology and tissue architecture. Computer assisted quantification of the staining intensity was performed on malignant and normal zones of human colon tissues to test the correlation between the immunolabelling signal and the respective histological patterns observed. RESULTS: Qualitative and quantitative differences were observed and measured between the normal and malignant part of each sample. Morphologically altered nuclei displayed densely labelled spots within faintly labelled areas whereas normal nuclei were darker and uniformly stained. Image analysis allowed calculation of the average integrated optical density of the nuclei in both types of tissues, demonstrating a constant and significantly lower intensity for the former type of cells.

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. Therefore an immunochemical technique was set up with the aim of demonstrating, in a population of mixed cells, the possibility of detecting the presence of individual nuclei containing hypomethylated DNA, on a cell-by-cell basis. Monoclonal antibodies to 5-methylcytidine were used to label cells grown in vitro. Under appropriate fixation and permeabilization conditions, interphase nuclei were labeled. Quantitative differences in the labeling were detected between Epstein-Barr virus-transformed cells and normal peripheral blood monocytes by flow cytometry analysis. Similar differences were observed by fluorescence microscopy. Both results were confirmed by Southern transfer and hybridization of DNA fragments generated by restriction enzyme digestion. This observation, which is in accordance with the occurrence of global DNA hypomethylation in tumors as established by chromatography, opens the field for the analysis of fresh tumor samples by flow cytometry and microscopy.

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. In this study we locked for cytological evidence that in the absence of an active poly(ADP-ribosyl)ation the DNA methylation pattern in L929 and NIH/3T3 mouse fibroblast cell lines is altered. For this purpose, differences in the methylation levels of interphase nuclei from control and treated cultures of two murine cell lines preincubated with 2 mM 3-aminobenzamide, an inhibitor of poly(ADP-ribosyl)ation, were measured in individual cells after indirect immunolabeling with anti-5MeC antibodies. The quantitative analysis allowed us to demonstrate that blocking of the poly(ADP-ribosyl)ation results in a higher number, size, and density of antibody binding regions in treated cells when compared to the controls. Analogously, sequential Giemsa staining and indirect immunolabeling of the same slides showed the heterochromatic regions colocalized with the extended methyl-rich domains.

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. 61:255-262, 1991) of control and 5-azacytidine-treated human cell cultures. A quantitative analysis of the number, total, and mean areas of labelled heterochromatic regions and the optical densities of euchromatin and heterochromatin was performed for the cells on microscope slides. Dedicated software was used to select and measure the areas of cytological interest. In additional experiments, DAPI-stained slides from control cultures were sequentially treated by in situ hybridization with beta-satellite DNA probe and indirect immunofluorescent labelling with anti-5MeC antibodies. Fluorescent signals of probe and antibodies were pseudocoloured and merged on digital images. The relative locations of probe- and antibody-positive areas were analysed on metaphases and nuclei, and their extensions were quantified in interphase nuclei. Our results show that (a) our analysis can successfully detect different levels of DNA methylation within individual nuclei, (b) in metaphase chromosomes the antibody binding sites are mostly coincident with the hybridisation sites, and (c) in interphase nuclei a quite different picture is consistently observed.

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. The proband's karyotype revealed the presence of a der(11) and two copies of a der(22), i.e. 47,XX,t(11;22)(q23;q11),+der(22) t(11;22)pat. The karyotype is the result of paternal 3:1 segregation after crossing-over involving the derived and the normal chromosome 22, as revealed by chromosome polymorphism analysis. Contrary to the preferential maternal, transmission of this common unbalanced translocation, the data from the literature, including our case, may suggest preferential paternal transmission of this rare type of unbalanced 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. 4-Chloro-1-naphthol (4C1N) was used as the substrate. The rate of methylation of individual chromosomes or chromosome groups was measured as the frequency of binding of anti-5MeC antibodies to specific chromosome regions. The following results were obtained: (i) in control cultures high intra- and interindividual variability in the binding frequencies of anti-5MeC antibodies to the short arm region of the acrocentrics was observed; (ii) 5-azaC consistently affects the methylation status of the constitutive heterochromatin; (iii) preferential demethylation occurs in the heterochromatic regions of specific chromosomes; (iv) under our experimental conditions the demethylating effect of 5-azaC appears not to be related to the well-known uncoiling effect of this drug.

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. Ribosomal gene activity was shown to be significantly increased by both agents. The increase persisted throughout the experiments, thereby suggesting the heritability of this epigenetic modification. The analysis of heritable DNA damage or modification is an important task in studying the risk of cancer onset and the mechanisms of cancer induction. In these studies two main results were obtained: (i) heritable DNA variations can be induced by both mutational and epigenetic changes; (ii) the modified end-point was not negatively selected.

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. In developing Xenopus laevis embryos at gastrulation, the bulk of the loops associated with histone-depleted nuclei exhibit a significant size increase, as visualized by fluorescence microscopy of the fully extended DNA halo surrounding high salt treated, ethidium bromide stained nuclei. This implies a reduction in the number of looped domains anchored to the supporting nucleoskeletal structure. The cytological analysis of metaphase plates from acetic acid fixed whole embryos, carried out in the absence of drugs inducing chromosome condensation, reveals a progressive thickening and shortening of metaphase chromosomes during development. We interpret these findings as a strong indication that the size and number of DNA loops influence the thickness and length of the chromosomes, respectively. The quantitative analysis of chromosome length distributions at different developmental stages suggests that the shortening is timed differently in different embryonic cells.

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. Exceptions were the two chromosomes 15 and chromosomes 13cp and 22p, all being strongly chromomycin-A3-positive in their short arms. These chromosomes bound to anti-5-MeC antibodies with differential frequencies in the different carriers. The possibility of involvement of repetitive GC-rich DNA in this behaviour is discussed.

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. One chromosome 21 had few gene copies and low activity. One chromosome 22 had many gene copies but low activity. Both chromosomes 14 had few gene copies but high activity. The level of expression that can be achieved by rRNA gene clusters can, therefore, be determined by factors other than the number of gene copies.

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). No response to GH administration was observed in lymphocytes from any of the three subjects with LD. These results suggest that the technique of selective silver staining of NOs can be usefully applied to the study of those growth disorders in which a peripheral unresponsiveness to GH is suspected, as demonstrated by data obtained on lymphocytes from patients with LD. This method seems to offer considerable potentialities for studying the cellular response also to other hormones and environmental stimuli.

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. The influence of different culture conditions on ribosomal gene responsiveness was also tested. Ribosomal gene activity was evaluated by selective silver staining of nucleolus organizing regions. The results show that hormone-induced enhancement of transcriptional activity requires both genetically determined cell responsiveness and environmentally determined permissive factors.

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. At this time, the number of benzidine-positive cells and the quantity of hemoglobin per cell are almost doubled. (b) Specific rRNA gene clusters are activated within single cells. Activation can be visualized at the single gene cluster level. (c) The increase in the average number of active ribosomal gene clusters per cell is not due to clonal selection, but rather to diffuse activation of several gene clusters. (d) The transcriptional activity of rRNA genes has been shown to be regulated at the cellular level by an agent known to specifically induce derepression of genes responsible for erythroid differentiation.

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. The observed differences became sharper in cells cultured under more restrictive conditions. Also, differences in the frequency of silver positivity of specific chromosomal NOs located on individually identified chromosomes were observed in cells from the same tissue. Furthermore, differences in the frequency of activation of rDNA clusters located on the same chromosome were also observed between cells from the two tissues. The possible biologic meanings of these findings are discussed.

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. A comparison with the late replication pattern of the normal X chromosome has also been done.