Active antineoplastic DNA intercalating agents such as m-AMSA produce breaks in the DNA of mammalian cells and their isolated nuclei. These cellular DNA breaks have been reproduced in an isolated chemical system consisting solely of DNA, intercalator and the purified nuclear enzyme, topoisomerase II (Topo II). Intercalators stabilize a protein-DNA complex which is an intermediate in the normal DNA breaking-rejoining cycle of Topo II. Thus the drugs inhibit the normal function of the enzyme. The purpose of this proposal is to investigate whether this enzyme inhibition is the mechanism by which intercalators kill malignant cells. Our previous work has suggested that intercalator-induced, DNA scission is highest in rapidly proliferating cells, cell populations enriched in the G1-S phase of the cell cycle or cells under hormonal stimulation to proliferate. We will extend these observations by examining m-AMSA-induced DNA scission in a number of cell systems: (1) quiescent vs. proliferating human fibroblasts (HF), (2) HeLa cells synchronized in various phases of the cell cycle, (3) epidermal growth factor (EGF)-stimulated HF, (4) EGF-treated human epidermoid carcinoma (A431) cells, (5) cells putatively deficient in Topo II and (6) an intercalator-sensitive/resistant cell pair. DNA filter elution will be used to quantify breakage. C14-m-AMSA transport and m-AMSA-induced cytotoxicity will also be quantified. Nuclear Topo will be isolated from these cells. Topo II will be purified by proven techniques and characterized using DNA agarose gel electrophoresis and a recently developed rapid filter-binding assay. We will focus particularly on the effects of m-AMSA on DNA cleaving, unwinding, catenating, and binding properties of the Topo from these cells to understand the biochemical basis for the differential cytotoxicity and DNA cleavage produced by m-AMSA. Topo II activity may also be found in the A431 membrane-localized EGF-receptor and will be similarly studied. This receptor has a high degree of homology with the v-erb-b oncogene product. Topo may be among the functions of a membrane receptor which is the product of an oncogene, and thus preferentially expressed in malignant cells. A novel mechanism of tumor-specific drug action may be the inhibition of "membrane-receptor Topo" by intercalating agents. The Topo II activity of isolated EGF receptor from A431 membranes will be compared with that from A431 nuclei.