Methylation levels of normal and chronic lymphocytic leukemia B lymphocytes: computer-assisted quantitative analysis of anti-5-methylcytosine antibody binding to individual nuclei

Capoa, A. de; Grappelli, C.; Febbo, F.R.; Spano, Anthony; Niveleau, Alain; Cafolla, Arturo; Cordone, Iole; Foà, Roberto

doi:10.1002/(sici)1097-0320(19990601)36:2<157::aid-cyto10>3.0.co;2-k

Cited by 8 publications

(2 citation statements)

References 14 publications

(10 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This feature was confirmed by image analysis performed on a series of 13 pairs of samples for which lower intensity staining of 17–29% was calculated in malignant samples compared with non-malignant counterparts. Similar results were obtained previously using the same technique for other types of malignant cells24 and cell lines 2325 26For colon adenocarcinomas, Feinberg and colleagues3detected 8–10% DNA hypomethylation after extraction of DNA, followed by nuclease digestion and high pressure liquid chromatography (HPLC) analysis.…”

Section: Discussionsupporting

confidence: 77%

See 1 more Smart Citation

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

Hernandez‐Blazquez

2000

Gut

View full text Add to dashboard Cite

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 paraYn 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. (Gut 2000;47:689-693)

show abstract

Section: Discussionsupporting

confidence: 77%

“…However, it is known that during cancer progression an increase in DNA methyltransferase activity is observed 3334 Simultaneously, large nuclear areas display a weak immunostaining signal which could reveal hypomethylated regions similar to those observed in leukaemic cells 24…”

Section: Discussionmentioning

confidence: 96%

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

Hernandez‐Blazquez

2000

Gut

View full text Add to dashboard Cite

show abstract

The Loss of Methyl Groups in DNA of Tumor Cells and Tissues

Niveleau

Piyathilake

Capoa³

et al. 2005

DNA Methylation and Cancer Therapy

View full text Add to dashboard Cite

Covisualization of Methylcytosine, Global DNA, and Protein Biomarkers for In Situ 3D DNA Methylation Phenotyping of Stem Cells

Tajbakhsh

2013

Imaging and Tracking Stem Cells

View full text Add to dashboard Cite

DNA methylation and histone modifications are key regulatory mechanisms in cellular differentiation, and are skewed in complex diseases. Therefore, analyzing the higher nuclear organization of methylated DNA in conjunction with relevant cellular components, such as protein biomarkers, may well add cell-by-cell-specific spatial and temporal information to quantitative molecular data for the discovery of stem cell differentiation-related signaling networks and their exploitation in the therapeutic reprogramming of cells. The in situ fluorescent covisualization of methylated DNA (methylated CG dinucleotides = MeC), global DNA (gDNA), and proteins has been challenging, as the immunofluorescence detection of MeC sites requires thorough denaturing of double-stranded DNA for antigen (methylated carbon-5 of cytosine) retrieval. The protocol we present overcomes this obstacle through optimization of cell membrane permeabilization, acid treatment, and intermediate fixation steps to preserve immunostaining of biomarkers and delineate MeC and gDNA, while conserving the captured three-dimensional (3D) structure of the cells; making it suitable for high-resolution confocal microscopy, 3D visualization, and topological analyses of fixed cultured cells as well as fresh and frozen tissue sections.

show abstract

Methylation levels of normal and chronic lymphocytic leukemia B lymphocytes: computer-assisted quantitative analysis of anti-5-methylcytosine antibody binding to individual nuclei

Cited by 8 publications

References 14 publications

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

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

The Loss of Methyl Groups in DNA of Tumor Cells and Tissues

Covisualization of Methylcytosine, Global DNA, and Protein Biomarkers for In Situ 3D DNA Methylation Phenotyping of Stem Cells

Contact Info

Product

Resources

About