DNA global hypomethylation in EBV-transformed interphase nuclei

In tumors, DNA is often globally hypomethylated compared to DNA extracted f rom normal tissues. This observation is usually made after extraction and e xhaustive digestion of DNA followed by analysis of nucleosides by chromatog raphy or digestion with restriction enzymes, gel analysis, and hybridizatio n. This approach provides an average value which does not give information on the various cell subpopulations included in heterogeneous samples. There fore an immunochemical technique was set up with the aim of demonstrating, in a population of mixed cells, the possibility of detecting the presence o f individual nuclei containing hypomethylated DNA, on a cell-by-cell basis. Monoclonal antibodies to 5-methylcytidine were used to label cells grown i n vitro. Under appropriate fixation and permeabilization conditions, interp hase nuclei were labeled. Quantitative differences in the labeling were det ected between Epstein-Barr virus-transformed cells and normal peripheral bl ood monocytes by flow cytometry analysis. Similar differences were observed by fluorescence microscopy. Both results were confirmed by Southern transf er and hybridization of DNA fragments generated by restriction enzyme diges tion. This observation, which is in accordance with the occurrence of globa l DNA hypomethylation in tumors as established by chromatography, opens the held for the analysis of fresh tumor samples by flow cytometry and microsc opy. (C) 1999 Academic Press.