Biochemical and proteomics approaches to characterize topoisomerase IIalpha cysteines and DNA as targets responsible for cisplatin-induced inhibition of topoisomerase IIalpha.

ABSTRACT

Cisplatin was shown to strongly inhibit the decatenation and relaxation activity of isolated human DNA topoisomerase IIalpha. This inhibition was not accompanied by stabilization of a covalent topoisomerase IIalpha-DNA intermediate. Pretreatment of kinetoplast plasmid DNA (kDNA) or pBR322 DNA with submicromolar concentrations of cisplatin quickly rendered these substrates incompetent in the topoisomerase IIalpha catalytic assay. Cisplatin nearly equally inhibited growth of a parental K562 and an etoposide-resistant K/VP.5 cell line that contained decreased topoisomerase IIalpha levels, a result consistent with isolated enzyme experiments demonstrating that cisplatin was not a topoisomerase IIalpha poison. Because cisplatin is known to react with protein sulfhydryl groups, the 13 cysteine groups in the topoisomerase IIalpha monomer were evaluated by mass spectrometry to determine which cysteines were free and disulfide-bonded to identify possible sites of cisplatin adduction. High-pressure liquid chromatography-matrix-assisted laser desorption ionization mass spectrometry showed that topoisomerase IIalpha contained at least five free cysteines (170, 216, 300, 392, and 405) and two disulfide-bonded cysteine pairs (427-455 and 997-1008). Cysteine 733 was also disulfide-bonded, but its partner cysteine could not be identified. Cisplatin antagonized the formation of a fluorescence adduct between topoisomerase IIalpha and the sulfhydryl-reactive maleimide reagent 10-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-naphtho[2,1-b]pyran-2-carboxylic acid methyl ester (ThioGlo-1). Dithiothreitol, which was shown by spectrophotometry to react rapidly with cisplatin (6-min half-time), diminished the capacity of cisplatin to interfere with ThioGlo-1 binding to topoisomerase IIalpha. The results of this study suggest that cisplatin may exert some of its cell growth inhibitory and antitumor activity by inhibition of topoisomerase IIalpha through reaction with critical enzyme sulfhydryl groups and/or by forming DNA adducts that render the DNA substrate refractory to topoisomerase IIalpha.