Bacteriophage T4 DNA topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in T4-infected Escherichia coli.

Abstract

The mammalian type II DNA topoisomerase has been proposed to be the intracellular target of a variety of antitumor agents, including m-AMSA [4'-(9-acridinylamino)-methanesulfon-m-anisidide]. Because the bacteriophage T4-encoded topoisomerase resembles the mammalian enzyme, we are using T4 as a simple model system to investigate the mechanism of action of m-AMSA. A mutation that renders T4 growth m-AMSA-resistant is closely linked to an amber mutation in T4 gene 39, which encodes one of the topoisomerase subunits. In addition, the gene 39 subunit from the m-AMSAresistant mutant phage has an altered net charge, strongly indicating that the drug-resistance mutation is within gene 39. Topoisomerase purified from mutant phage-infected Escherichia coli exhibits drug-insensitive DNA relaxation and DNA cleavage activities. Because a single mutation results in both drug-resistant phage growth and a drug-insensitive viral topoisomerase, we conclude that the T4-encoded type II DNA topoisomerase is the physiological target of m-AMSA in phageinfected E. coli.The mammalian type II DNA topoisomerase has been proposed to be the intracellular target of several classes of antitumor agents, including acridine derivatives, anthracyclines, and certain epipodophyllotoxins (for review, see ref.1). These agents block topoisomerase activity in vitro and cause the accumulation of covalent complexes consisting of topoisomerase attached to each 5' terminus of double-strand DNA breaks (2, 3). Because the type II topoisomerase catalyzes passage of one duplex DNA segment through a transient double-strand break in a second DNA segment, the drug-induced covalent complexes are thought to be identical to an intermediate in the normal strand-passage reaction (3). The accumulation of covalent complexes in the presence of these agents suggests that the drugs block the resealing step of the topoisomerase reaction cycle.Although it is clear that topoisomerase is inhibited by these various antitumor agents, a definitive assignment of the specific intracellular target has not been made. Some of these agents inhibit other important cellular enzymes, such as DNA polymerase (4,5). In addition, the quantity of protein-linked DNA breaks does not always correlate with cytotoxicity (6, 7), and protein-free DNA breaks have been detected in drug-treated cells (4, 7). Although studies using drugresistant mammalian cell lines are consistent with the model that topoisomerase is the target of drug action (8-12), in no case has a single mutation been shown to confer both drug-resistant cell growth and drug-resistant topoisomerase activity.To circumvent problems encountered in complex mammalian systems, we used the simple bacteriophage T4 (13) as a model system to investigate the mechanism of action of the antitumor agent 4'-(9-acridinylamino)methanesulfon-manisidide (m-AMSA). Many of the properties of the T4 topoisomerase are similar to those of the mammalian, but not the bacterial, type II topoisomerase (14, 15). Most importantly, both the mammalian and phag...

An Antitumor Drug-Induced Topoisomerase Cleavage Complex Blocks a Bacteriophage T4 Replication Fork In Vivo

“…The antitumor drug m-AMSA blocks T4 growth by targeting the phage-encoded type II topoisomerase (31). Nevertheless, this enzyme is not essential for T4 DNA replication, presumably because the host DNA gyrase can substitute for it (45).…”

“…Phage T4 encodes a type II DNA topoisomerase that is inhibited by many of the same antitumor drugs that block the mammalian enzyme. The T4 topoisomerase was demonstrated to be the physiological target for m-AMSA (31), and inhibition of phage growth is probably dependent on cleavage complex formation (see above) (50). Recombinational repair of damage derived from the drug-induced cleavage complex is important in both the bacteriophage T4 and mammalian systems, which also argues that T4 is a useful model system for these studies.…”

Mechanisms of Multidrug Resistance in Cancer Treatment

“…A new restriction enzyme polymorphism was found in the resistant forms. A mutation at the ATP binding site has been found to induce mAMSA resistance in the bacteriophage T4 DNA topoisomerase (42,43). The extent to which downregulation of the total amount of top0 I1 is a significant resistance mechanism remains unclear, although reduced levels of top0 I1 shown in some studies could be due to decreased half-life of the mutant protein.…”

Disruption of a topoisomerase-DNA cleavage complex by a DNA helicase.

“…Most importantly, both are sensitive to the aminoacridine m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide] (10, 11). A point mutation in one of the genes encoding the bacteriophage T4 topoisomerase allows drug-resistant phage growth, and topoisomerase purified from the mutant phage has drug-resistant topoisomerase activity (12,13).Topoisomerase inhibitors convert the enzymes that they target into poisons by inducing formation of the cleavage complex, a form of DNA damage. This view was first articulated when it was found that bacteriophage 17 …”

“…Most importantly, both are sensitive to the aminoacridine m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide] (10,11). A point mutation in one of the genes encoding the bacteriophage T4 topoisomerase allows drug-resistant phage growth, and topoisomerase purified from the mutant phage has drug-resistant topoisomerase activity (12,13).…”