Three distinct DNA topoisomerases have been identified in human nuclei: DNA topoisomerase (Topo I) and two isozymes (alpha and beta) of DNA topoisomerase II (Topo II). These enzymes are targets of several classes of antitumor drugs, but little is known about their levels in human tumor cells. We have developed an immunohistochemical method to measure the levels of these enzymes in fixed tissues, and our preliminary results indicate that both isozymes of Topo II are elevated in most breast carcinomas. We propose to extend these results in a much larger study by measuring the abundance of Topo II by automated immunohistochemistry using monoclonal antibodies against tumor antigens and oncoproteins. The results will be quantified by image analysis. These results will be compared to immunocytochemical measurements of the levels of Topo II in MCF7 cells at various times after treatment with estradiol. The immunocytochemical measurements will also be compared to catalytic activity assays of DNA topoisomerases performed with nuclear extracts prepared from MCF7 cells and selected specimens of fresh tumors. Topo I activity will be determined by relaxation of supercoiling of plasmid pHC624 DNA, and Topo II activity will be measured by unknotting of bacteriophage P4 DNA. The abundance of mRNAs encoding Topo II will be measured by in situ hybridization of mRNA in fresh tissues and preparations of HuT 78 cells to probes specific for the three genes. These studies will compare the levels of protein and mRNA for all three enzymes in individual cells. The proposed research will compare and contrast the levels of mRNA and protein for the three DNA topoisomerases in stages I-IV breast carcinomas. Furthermore, the levels of these enzymes will be correlated to the clinical response of the tumors to topotecan and doxorubicin, agents that inhibit Topo I and/or Topo II. If a positive correlation is observed, then it would be highly desirable to routinely screen breast carcinomas for levels of Topo II by the methods that we have developed prior to chemotherapy.
