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DNA methylation and tumor progression in normal, dysplastic and neoplastic cells from uterine cervixa Dept Genet and Mol Biol, Univ Rome1, Italy; b Pathology, and c Gynecol Oncol Dept, Polo Oncologico Rome, Italy; d Dept. Biotech Hematol, Univ. Rome1, Italy; e Lab.Anatomopathologie, Hopital Herriot, Lyon; f Univ. Fourier, Grenoble, France.AIM: Analysis of the extent of DNA methylation in: 1-individual nuclei and, 2-tissue sections from normal, dysplastic and tumor samples (CIN1,CIN2 and cancer) of the uterine cervix by computer-assisted quantitative analysis. METHODS: 1-Indirect immunolabeling by anti-5-MeC-antibodies was performed on cytological smears. The extent of eu-and heterochromatin DNA methylation within each nucleus was quantitated on a cell-by-cell basis. Additional slides from these samples were stained by Papanicolaou for cytological diagnosis. 2-Paraffin-embedded tissue sections from controls and pathological patients were indirectly immunolabeled by anti-5-MeC antibodies. In each section the mean optical density of nuclei was quantified. In all experiments computer-assisted quantitative analyses of methylation levels were performed by dedicated softwares. RESULTS In smears of individual nuclei from normal vs. HSIL patients significant differences in the methylation levels as for the number, size and optical density of heterochromatic regions were observed. Analogously, the nuclei of invasive carcinomas with different degrees of malignancy were significantly different from the HSIL nuclei. Similar results were obtained on tissue sections, the ratio between the density of pathological and normal tissues -assuming a 1.0 value for normal tissues- being 0.52, 0.49 and 0.58 for CIN1, CIN2 and cancer patients respectively. CONCLUSIONS The deregulation of DNA methylation is known to be strictly related to tumorigenesis. In our experiments it was consistently observed that the earliest intranuclear modifications occurring in the switch from normal to dysplastic and cancer nuclei were the progressively decreasing numbers and sizes of the 5-methylcytosine-rich heterochromatic regions. In more advanced stages of tumor evolution, progressively decreasing levels of nuclear eu- and heterochromatin DNA methylation and progressive enlargement of nuclear size were consistently observed in cancer cells. KEY WORDS: Cervix cancer, DNA methylation, anti-5MeC antibodies. For more information, contact adriana.decapoa@uniroma1.it Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Chromatin Structures. |
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