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Journal of Histochemistry and Cytochemistry, Vol. 45, 251-264, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Detection of Human Topoisomerase II{alpha} in Cell Lines and Tissues: Characterization of Five Novel Monoclonal Antibodies

Udo Kellnera, Hans-Juergen Heidebrechta, Pierre Rudolpha, Harald Biersackc, Friedrich Buckd, Thomas Dakowskia, Hans-Heinrich Wackerb, Marcus Domanowskia, André Seidela, Ole Westergaardc, and Reza Parwareschb
a Department of General Pathology, University of Kiel, Kiel, Germany
b Department of Hematopathology, University of Kiel, Kiel, Germany
c Department of Molecular and Structural Biology, University of Aarhus, Aarhus, Denmark
d Institute of Cell Biochemistry and Clinical Neurobiology, University of Hamburg, Hamburg, Germany

Correspondence to: Udo Kellner, Dept. of General Pathology, University of Kiel, Michaelisstr. 11, 24105 Kiel, Germany.

We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the {alpha}-isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase IIß was ruled out by testing the antibodies on crude extracts from yeast cells expressing the ß-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology. (J Histochem Cytochem 45:251-263, 1997)

Key Words: human topoisomerase II{alpha}, monoclonal antibodies, Western blot analysis, immunohistochemistry, confocal laser microscopy, proliferation


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