The effect of antitumor epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), on chromosomal DNA in mammalian cells was studied using SV40 virus-infected monkey cells as a model system. Treatment of SV40 virus-infected monkey cells with these drugs results in DNA breaks on intracellular SV40 DNA. The broken DNA strands are sensitive to phenol extraction, suggesting that they are associated with tightly linked protein(s). Several pieces of evidence suggest that DNA topoisomerase II is covalently linked to the broken SV40 DNA strands following drug treatment. (a) Novobiocin, an inhibitor of topoisomerase II, blocks the epipodophyllotoxin-induced SV40 DNA breaks in vivo and in vitro. (b) Epipodophyllotoxin-induced cleavage sites on intracellular SV40 DNA are strikingly similar to those produced on purified SV40 DNA by purified calf thymus DNA topoisomerase II. (c) The protein-linked SV40 DNA is specifically immunoprecipitated by antisera against topoisomerase II. We thus conclude that epipodophyllotoxins induce chromosomal DNA breakage via DNA topoisomerase II. The physiological effects of epipodophyllotoxins on cell death, chromosomal DNA breakage, sister chromatid exchanges, and chromosomal aberrations may be the consequence of drug interaction with DNA topoisomerase II. Our present results are also consistent with the proposal that epipodophyllotoxins interfere with the breakage-reunion reaction of DNA topoisomerase II by stabilizing an enzyme-DNA complex in its putative cleavable state.
This work has been supported by NIH Grants GM-27731 and CA-99632.