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Received for publication July 2, 2004.
Revised November 10, 2004.
Accepted for publication December 14, 2004.
Cysteines and DNA as Targets Responsible for Cisplatin-induced Inhibition of Topoisomerase II
Cisplatin was shown to strongly inhibit the decatenation and relaxation activity of isolated human DNA topoisomerase II
. This inhibition was not accompanied by stabilization of a covalent topoisomerase II-DNA intermediate. Pretreatment of kDNA or pBR322 DNA with sub-micromolar concentrations of cisplatin quickly rendered these substrates incompetent in the topoisomerase II catalytic assay. Cisplatin nearly equally inhibited growth of a parental K562 and an etoposide-resistant K/VP.5 cell line that contained decreased topoisomerase II levels, a result consistent with isolated enzyme experiments demonstrating that cisplatin was not a topoisomerase II poison. Because cisplatin is known to react with protein sulfhydryl groups, the 13 cysteine groups in the topoisomerase II monomer were evaluated by mass spectrometry to determine which cysteines were free and disulfide-bonded in order to identify possible sites of cisplatin adduction. HPLC-MALDI mass spectrometry showed that topoisomerase II contained at least five free cysteines (170, 216, 300, 392, 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 II and the sulfhydryl-reactive maleimide reagent, 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 II. The results of this study suggest that cisplatin may exert some of its cell growth inhibitory and antitumor activity by inhibition of topoisomerase II through reaction with critical enzyme sulfhydryl groups and/or by forming DNA adducts that render the DNA substrate refractory to topoisomerase .
Key words:
Mass Spectroscopy, Mechanisms of cell killing/apoptosis, Topoisomerases
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