203 Molecular and Cellular Biochemistry 178: 203–212, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands. Inhibition of human topoisomerase II by anti- neoplastic benzazolo[3,2- a]quinolinium chlorides Pablo E. Vivas-Mejía, 1,2 Osvaldo Cox 1 and Fernando A. González 1,2 1 Department of Chemistry, 2 Biotesting Center, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico Received 12 February 1997; accepted 15 July 1997 Abstract Previously we reported [20] that there is no correlation between the cytotoxic activity of four new structural analogs of the antitumor DNA intercalator 3-nitrobenzothiazolo[3,2-a]quinolinium chloride (NBQ-2) and their interaction with DNA. In the present study, we present evidence suggesting that the molecular basis for the anti-proliferative activity of these drugs is the inhibition of topoisomerase II. The NBQ-2 derivatives inhibited the relaxation of supercoiled DNA plasmid pRYG mediated by purified human topoisomerase II. Inhibition of the decatenation of kinetoplast DNA mediated by partially purified topoisomerase II extracted from the human histiocytic lymphoma U937 (a cell line previously shown to be sensitive to the drugs) was also caused by these drugs. The potency of the benzazolo[3,2-a]quinolinium drugs against topoisomerase II in vitro was the following: 7- (1-propenyl)-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-59) > 4-chlorobenzothiazolo[3,2-a]quinolinium chloride (NBQ- 76) > 7-ethyl-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-48) > 7-benzyl-3-nitrobenzimidazolol[3,2-a]quinolinium chloride (NBQ-38). This rank of potency for topoisomerase II inhibition correlated very well with the cytotoxicity elicited by these drugs. Furthermore, significant levels of topoisomerase II/DNA cleavage complex induced by these drugs in vivo were detected when U937 cells were treated with NBQ-59 and NBQ-76 whereas NBQ-38 and NBQ-48 produced negligible amounts of the cleavage complex. Our results strongly suggest that topoisomerase II is the major cellular target of this family of compounds. (Mol Cell Biochem 178: 203–212, 1998) Key words: antitumor, DNA intercalator, cancer Address for offprints: F.A. González, Department of Chemistry and Biotesting Center, University of Puerto Rico, Rio Piedras Campus, P.O. Box 23346, San Juan, Puerto Rico 00931-3346 Introduction In order to maintain the flow of genetic information, DNA must be able to disrupt and reform its structure throughout the cell cycle. Different enzymes are associated with these processes; among them are topoisomerases I and II [1]. These enzymes mediate the transient breakage of a single strand of DNA (topoisomerase I), or of both strands of DNA (topoisomerase II), allowing strands to pass through the nick, and then rejoining them with a concomitant change in superhelicity [2, 3]. These nuclear enzymes are responsible for controlling, maintaining and modifying the structure or topology of DNA during replication, transcription and recombination [4, 5]. In addition, topoisomerase II has been shown to participate in mitotic chromosome condensation [6], and in the decatenation of intertwined daughter chromo- somes at the end of DNA replication [7, 8]. As a consequence of the involvement of topoisomerase II in these essential cellular processes, this enzyme is a cellular target for several clinically active anticancer drugs [9, 10]. Most of the well-characterized topoisomerase II inhibi- tors interact directly with these cleavage/religation pro- cesses by trapping topoisomerase II on DNA in a covalently bound state, often referred to as the ‘cleavage complex’ [11]. Among the drugs that stabilize this reaction intermediate are the non-DNA-intercalating epipodophilotoxins, etoposide (VP-16) and teniposide (VM-26) [12, 13]. However, not all topoisomerase II inhibitors are non-intercalating agents. The most widely used intercalating inhibitors (such as 4′-(9- acridinylamino)-methanesulfon-m-aniside (m-AMSA), ellipticine and adriamycin) also act by inducing the cleavage complex formation [14–16].