Immunohistochemical detection of acetylation and phosphorylation of histone H3 in cervical smears
Department of Experimental Oncology, Masaryk Cancer Institute, Žlutý kopec 7, Czech Republic
AIM: Histones represent a perspecive marker based on the detection of posttranslationally modified nuclear proteins. Histone H3 modifications (phosphorylation and acetylation) were detected in cervical smears and correlated with histology. METHODS: Liquid based cytologic specimens were obtained from 56 women before conisation. The immunohistochemical procedure was performed using peroxidase-labeled avidin-biotin system. Samples were stained with polyclonal serum against unmodified peptide of human histone H3. Samples were incubated with the primary antibodies overnight at 4°C in humidity chambers at the dilutions 1:2000 and 1:5000. Anti-rabbit IgG biotin conjugate was used as secondary antibody at 1:50 dilution. The immunopositivity of cells (200 randomly selected nuclei) was examined under light microscope. RESULTS: Immunohistochemical detection of posttranlationally modified histone H3 showed nuclear positivity for phosphorylated form of histone H3. For acetylated form of histone H3 we revealed a remarkable granular appearance of positive nuclei. Nuclear positivity for phosphorylated form (P) and acetylated form (A) of H3 in CIN II(23%P, 33%A) and CIN III (23%P, 44%A)was higher in comparision with metaplasia (11%P, 12%A) and CIN I(8%P, 15%A). CONCLUSION: We revealed a marked association of histone H3 modifications with the progression of CIN II and CIN III in comparision to metaplasia and CIN I. Staining of cells with anti-phospho-histone H3 antibodies provides a highly specific marker for mitosis. Acetylation of nucleosomal histones correlates with localised transcriptional acivity.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 897 (Precursor lesions).