A number of clinically important anticancer agents including the epipodophyllotoxins (VP-16, VM-26), aminoacridines (m-AMSA), anthracyclines (doxorubicin, daunorubicin), and anthracenediones (mitoxantrone), are known to interfere with DNA topoisomerase II activity. However, large gaps persist in our knowledge concerning the critical biochemical events following inhibition topoisomerase II activity that lead to blockade of tumor cell proliferation. DNA topoisomerase II is a major nonhistone protein of the nuclear matrix. Accordingly, the overall goal of the proposed continuation studies is to provide insights into the mechanisms by which drug-stabilized topoisomerase II-DNA complexes interfere with the nuclear matrix functions of DNA organization and replication. The proposed studies will extend our ongoing work and will utilize new techniques for investigating drug interactions at DNA replication forks associated with the nuclear matrix. The specific objectives of the initial studies are to determine a) if topoisomerase II-DNA complexes stabilized by either VM-26 or m-AMSA are enriched in nuclear matrix DNA compared to bulk DNA, and b) to what extent the alpha and beta isoforms of topoisomerase II are involved in cleavable complex formation with nuclear matrix and bulk DNA. These studies will be carried out with parental CCRF-CEM cells and with the CEM/VM-l subline that is resistant to multiple topoisomerase II-active agents. The results obtained will provide insights into the importance of the individual topoisomerase II isoforms in DNA replication and as targets for topoisomerase II-active agents. Further studies will involve testing the working hypothesis that drug-stabilized topoisomerase II-DNA complexes act as physical barriers to the movement of DNA replication complexes along the template DNA. This will be accomplished by monitoring the effects of VM-26 and m-AMSA on the movement of DNA replication forks through the c-myc protooncogene and the dihydrofolate gene. A second working hypothesis to be tested is that cleavable complex formation in DNA replication forks induced by either VM-26 or m-AMSA results in the detachment of the DNA replication forks from the nuclear matrix. A direct relationship between the extent of drug-induced cleavable complex formation in nuclear matrix DNA and the extent of dissociation of Okazaki fragments from the nuclear matrix would provide evidence that DNA strand breaks at replication forks are critical lesions induced by these topoisomerase II-active agents.
