Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II.

Adriamycin (doxorubicin), a potent antitumor drug in clinical use, interacts with nucleic acids and cell membranes, but the molecular basis for its antitumor activity is unknown. Similar to a number of intercalative antitumor drugs and nonintercalative epipodophyllotoxins (VP-16 and VM-26), adriamycin has been shown to induce single- and double-strand breaks in DNA. These strand breaks are unusual because a covalently bound protein appears to be associated with each broken phosphodiester bond. In studies in vitro, mammalian DNA topoisomerase II mediates DNA damage by adriamycin and other related antitumor drugs.

Mechanism of antitumor drug action: poisoning of mammalian DNA topoisomerase II on DNA by 4'-(9-acridinylamino)-methanesulfon-m-anisidide.

The intercalative acridine derivative 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), but not its isomer o-AMSA, is a potent antitumor drug that in mammalian cells stimulates the formation of DNA strand breaks that are characterized by tightly bound proteins. Using purified mammalian DNA topoisomerases, we have analyzed the effects of these antitumor drugs on topoisomerase-DNA interactions. The antitumor drug m-AMSA dramatically stimulates the formation of a topoisomerase II-DNA complex that is detected on protein-denaturant treatment: both single- and double-stranded DNA breaks are produced and a topoisomerase II subunit is linked covalently to each 5' end of the broken DNA strands. The noncytotoxic isomer, o-AMSA, which does not induce significant amounts of DNA breaks in cultured cells, exhibits a correspondingly smaller effect in stimulating formation of the complex in vitro. The agreement between in vitro and in vivo studies suggests that mammalian DNA topoisomerase II may be the primary target of m-AMSA and that the drug-induced complex formation between topoisomerase II and DNA may be the cause of cytotoxicity and other effects such as DNA sequence rearrangements and sister-chromatid exchange.

Identification of the breakage-reunion subunit of T4 DNA topoisomerase.

The antitumor drug 4'-(9-acridinylamino)methanesulfon-m-anisidide which stimulates the cleavable complex formation between mammalian DNA topoisomerase II and DNA also stimulates the cleavable complex formation between bacteriophage T4-induced DNA topoisomerase and DNA. In the presence of 4'-(9-acridinylamino)methanesulfon-m-anisidide, T4 DNA topoisomerase and DNA form a "cleavable complex" which is characterized by its sensitivity to protein-denaturant treatment. Upon protein-denaturant treatment, the phosphodiester bond of DNA is cleaved, and the gene 52 protein subunit of the topoisomerase becomes covalently linked to the 5'-end of the broken DNA. The covalent protein-DNA linkage has been determined by both paper electrophoresis and thin layer chromatography to be tyrosyl phosphate.

Cleavage of DNA by mammalian DNA topoisomerase II.

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.

Intercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II.

Many intercalative antitumor drugs have been shown to induce reversible protein-linked DNA breaks in cultured mammalian cells. Using purified mammalian DNA topoisomerase II, we have demonstrated that the antitumor drugs ellipticine and 2-methyl-9-hydroxyellipticine (2-Me-9-OH-E+) can produce reversible protein-linked DNA breaks in vitro. 2-Me-9-OH-E+ which is more cytotoxic toward L1210 cells and more active against experimental tumors than ellipticine is also more effective in stimulating DNA cleavage in vitro. Similar to the effect of 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) on topoisomerase II in vitro, the mechanism of DNA breakage induced by ellipticines is most likely due to the drug stabilization of a cleavable complex formed between topoisomerase II and DNA. Protein denaturant treatment of the cleavable complex results in DNA breakage and covalent linking of one topoisomerase II subunit to each 5'-end of the cleaved DNA. Cleavage sites on pBR322 DNA produced by ellipticine or 2-Me-9-OH-E+ treatment mapped at the same positions. However, many of these cleavage sites are distinctly different from those produced by the antitumor drug m-AMSA which also targets at topoisomerase II. Our results thus suggest that although mammalian DNA topoisomerase II may be a common target of these antitumor drugs, drug-DNA-topoisomerase interactions for different antitumor drugs may be different.

Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II.

Many intercalative antitumor drugs have been shown to cleave DNA indirectly through their specific effect on the stabilization of a cleavable complex formed between mammalian DNA topoisomerase II and DNA (Nelson, E.M., Tewey, K.M., and Liu, L.F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1361-1365). Antitumor epipodophyllotoxins (VP-16 and VM-26) which do not intercalate DNA can similarly induce protein-linked DNA breaks in cultured mammalian cells. In vitro studies using purified mammalian DNA topoisomerase II show that epipodophyllotoxins interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by stabilizing a cleavable complex. Treatment of this stabilized cleavable complex with protein denaturants results in DNA strand breaks and the covalent linking of a topoisomerase subunit to the 5'-end of the broken DNA. Furthermore, epipodophyllotoxins also inhibit the strand-passing activity of mammalian DNA topoisomerase II, presumably as a result of drug-enzyme interaction. The agreement between the in vivo and in vitro studies suggests that mammalian DNA topoisomerase II is a drug target in vivo. The similarity between the effect of epipodophyllotoxins on mammalian DNA topoisomerase II and the effect of nalidixic acid on Escherichia coli DNA gyrase suggests that the cytotoxic action of epipodophyllotoxins may be analogous to the bactericidal action of nalidixic acid.

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22.

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.