Localization of Anti-Topoisomerase IIa Antibodies under Normal Conditions and after Artificial Chromosome Condensation and Decondensation

摘要

By means of immunofluorescence method, localization of DNA-topoisomerase IIα (Topo IIα) ininterphase nuclei and chromosomes at different stages of mitosis was studied in situ under normal conditionsand after treatment with condensing and decondensing solutions. In non-isolated mitotic M-HeLa cell chromo-somes, Topo IIα was uniformly distributed along chromatids after fixation and permeabilization in situ. Aftertreatment of cells with decondensing solutions (10 mM Tris; 0.1 mM CaCl_2in 10 mM Tris; 0.3 CaCl_2in10 mM Tris; 15% DMEM; 75 mM KC1), Topo Ha was evenly distributed along chromatids in prophase,prometaphase and metaphase; its concentration was the highest in the pericentromere region. After treatmentof cells with condensing solutions containing 0.7 mM, 1 mM, 2 mM or 3 mM CaCl_2in 10 mM Tris, Topo IIαwas not detected in prophase, metaphase and anaphase. However, in late telophase anti-Topo IIα antibodieswere found in reforming nuclei under identical conditions. After sequential treatment with condensing anddecondensing solutions, the distribution patterns of Topo Ha in chromosomes were the same as after treatmentwith only decondensing solutions. In anaphase and telophase, Topo IIα was evenly distributed along chroma-tids, while in prophase, prometaphase and metaphase it was predominantly localized in the pericentromereregion. After the treatment of cells with condensing solutions chromosome staining was not observed, appar-ently due to "masking" of binding sites for anti-Topo IIα antibodies. Homogenous distribution of Topo IIαalong chromatids in non-isolated chromosomes was preserved after the treatment of cells with hypotonic solu-tions; however, under these conditions Topo IIα concentration was higher in centromeres.