On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex

Abstract

Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.

Kinetics and Mechanisms of Activation of α-Amino Acid Ester Prodrugs of Camptothecins

“…The crude product was purified by column chromatography on silica gel (CH 2 Cl 2 -CH 3 COCH 3 , 95:5, v/v) to afford the title compound 7 (92 mg, 84%) as a yellow solid. 1 …”

DNA Damage Induces the Accumulation of Tiam1 by Blocking β-TrCP-dependent Degradation

“…It is plausible that cell type and DNA damage drugs are key determinants for the function of Tiam1 in apoptosis. Camptothecin (CPT) and doxorubicin (DOX), which target DNA topoisomerase I and II, respectively, are both anticancer drugs (25,26). Understanding the mechanisms that determine the cell response to these drugs is of high value for cancer treatment in the clinic.…”

A phase I study with MAG-camptothecin intravenously administered weekly for 3 weeks in a 4-week cycle in adult patients with solid tumours

“…The action of CPT is most evident in the S-phase of the cell cycle; therefore, prolonged inhibition of topoisomerase I was postulated to be an important parameter in causing cytotoxicity (Hertzberg et al, 1989;Del Bino et al, 1991;Kingsbury et al, 1991;Caiolfa et al, 2000). The relation between bladder toxicity and the dose excreted in 24 h suggests that bladder toxicity may be explained by interindividual differences in pharmacokinetics.…”