Localization of anti-topoisomerase IIα antibodies under normal conditions and after artificial chromosome condensation and decondensation

Abstract

By means of immunofluorescence method, localization of DNA-topoisomerase IIα (Topo IIα) in interphase nuclei and chromosomes at different stages of mitosis was studied in situ under normal conditions and after treatment with condensing and decondensing solutions. In non-isolated mitotic M-HeLa cell chromosomes, Topo IIα was uniformly distributed along chromatids after fixation and permeabilization in situ. After treatment of cells with decondensing solutions (10 mM Tris; 0.1 mM CaCl2 in 10 mM Tris; 0.3 mM CaCl2 in 10 mM Tris; 15% DMEM; 75 mM KCl), Topo IIα was evenly distributed along chromatids in prophase, prometaphase and metaphase; its concentration was the highest in the pericentromere region. After treatment of cells with condensing solutions containing 0.7 mM, 1 mM, 2 mM or 3 mM CaCl2 in 10 mM Tris, Topo IIα was not detected in prophase, metaphase and anaphase. However, in late telophase anti-Topo IIα antibodies were found in reforming nuclei under identical conditions. After sequential treatment with condensing and decondensing solutions, the distribution patterns of Topo IIα in chromosomes were the same as after treatment with only decondensing solutions. In anaphase and telophase, Topo IIα was evenly distributed along chromatids, while in prophase, prometaphase and metaphase it was predominantly localized in the pericentromere region. After the treatment of cells with condensing solutions chromosome staining was not observed, apparently due to “masking” of binding sites for anti-Topo IIα antibodies. Homogenous distribution of Topo IIα along chromatids in non-isolated chromosomes was preserved after the treatment of cells with hypotonic solutions; however, under these conditions Topo IIα concentration was higher in centromeres.