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 tu mours. This alteration is detected in early adenomas in colorectal tumorige nesis. Information is currently acquired after extraction of DNA from tissu es, digestion with nucleases, and analysis by reverse phase chromatography, or treatment with restriction enzymes followed by gel electrophoresis anal ysis 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 fr om 13 patients undergoing surgical resection of colorectal adenocarcinomas. Methods-Antibodies raised against 5-methylcytidine can be used to label met hyl rich regions in interphase nuclei. This technique was adapted to the st udy of paraffin embedded tissues and an immunohistochemical method was deve loped to assess the global methylation status of individual nuclei while pr eserving cell morphology and tissue architecture. Computer assisted quantif ication of the staining intensity was performed on malignant and normal zon es of human colon tissues to test the correlation between the immunolabelli ng 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 alte red nuclei displayed densely labelled spots within faintly labelled areas w hereas normal nuclei were darker and uniformly stained. Image analysis allo wed calculation of the average integrated optical density of the nuclei in both types of tissues, demonstrating a constant and significantly lower int ensity for the former type of cells.