Abstract
Bulgarein, a fungal metabolite, induced mammalian topoisomerase I-mediated DNA cleavage in vitro. The cleavage activity of bulgarein was comparable to that of camptothecin at a drug concentration range of 0.025-approximately 5 microM. The DNA cleavage induced by bulgarein was suppressed at concentrations above 12.5 microM. Treatment of a reaction mixture containing bulgarein and topoisomerase I with elevated temperature (65 degrees C) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I-mediated DNA cleavage induced by bulgarein is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex." Intensity of cleaved DNA fragments induced by bulgarein with topoisomerase I was different from that induced by camptothecin. Bulgarein inhibited catalytic activities of both topoisomerase I and topoisomerase II. The changes in absorption spectra of bulgarein in the visible region observed upon addition of increasing amounts of calf thymus DNA indicate that bulgarein interacts with DNA. DNA (un)winding assay by two-dimensional gel electrophoresis showed that bulgarein induced the winding of DNA in the opposite direction to that of an intercalator so that positively supercoiled molecules are produced. Thus, bulgarein represents a new class of drugs which stabilizes the cleavable complex of topoisomerase I and alters the structure of DNA in a manner leading to a tightening of the helical twist.
Highlights
Drugs that have been shown to stabilize the cleavable complex with topoisomerase may be classified into two groups
Bulgarein in the visible region observed upon addition 26) and the terpentecin family (UCT 4B, terpentecin,and of increasing amounts of calf thymus DNA indicate clerocidin) as topoisomerase I1 poisons [9, 11, 13]
Bulgarein represents a new class of drugs which stabilizes the cleavable complex of topoisomerase I and alters the structure of DNA in a manner leading to a tightening of the helical twist
Summary
Drugs that have been shown to stabilize the cleavable complex with topoisomerase may be classified into two groups. Inpanel A, 0.45 pg of supercoiled DNA was incubated with 20 units of topoisomerase I in the presence of drugs (lanes c-p) followedby SDS/proteinase K treatment and thenanalyzed on an agarose gel containing 0.5 pg/ml ethidium bromide.
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