RESUMEN
A computer-assisted assay based on the quantitative analysis of DNA methylation in individual interphase nuclei by indirect immunolabelling with anti-5-methylcytosine antibodies was recently developed in our laboratory. In situ analyses were performed on individual nuclei from normal and experimentally hypo- or hypermethylated cultured cells as well as on human peripheral blood B-lymphocytes from normal and chronic lymphoid leukemia (CLL) samples. We present the results obtained on cells from patients affected by different degrees of preneoplastic or neoplastic changes of the uterine cervix as compared to normal controls. The analysis of DNA methylation in individual cells from cytofuge samples was performed as follows: within each nucleus the eu- and heterochromatin methylation levels were quantified in the grey scale range by dedicated software in terms of numbers, areas and optical densities (ODs) of the immunolabeled dense heterochromatic regions ("spots"), and of the optical density of nuclear background, i.e., of nuclear euchromatin. Analogously, in randomly chosen microscope fields of tissue sections from paraffin-embedded samples, progressive tissue demethylation was observed in dysplastic and cancer cells as compared to normal ones. Both methods showed significant and progressive DNA hypomethylation in dysplastic and cancer cells as compared to control specimens.
Asunto(s)
5-Metilcitosina/análisis , Metilación de ADN , ADN de Neoplasias/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Lesiones Precancerosas/genética , Neoplasias Uterinas/genética , 5-Metilcitosina/inmunología , Adulto , Anticuerpos Monoclonales/análisis , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular , Cuello del Útero/metabolismo , Cuello del Útero/patología , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Hibridación Fluorescente in Situ , Persona de Mediana Edad , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
BACKGROUND: Most cancers show abnormal DNA methylation and a positive correlation between hypomethylation and tumour progression. PATIENTS AND METHODS: In our laboratory the extent of DNA methylation in individual nuclei in normal, cancer and non-cancer thyroid tissue samples was quantified according to a previously described method of computer-assisted semi-quantitative analysis. Cancer and non-cancer samples were obtained from nine patients with different thyroid pathologies (one multinodular goitre and eight carcinomas). Quantitative analysis was performed in two sets of samples, i.e. individual nuclei from touch preparations and from tissue sections. RESULTS: In all cancer specimens a statistically significant decrease of heterochromatin methylation was consistently observed. In both sets of samples a direct correlation was consistently observed between the extent of chromatin demethylation and the degree of malignancy. CONCLUSION: Our preliminary results suggest that our method of cell-by-cell detection of intranuclear methylation abnormalities may be a useful tool in early identification of thyroid cancer lesions.