Murine Transgenic Cells Lacking DNA Topoisomerase IIb Are Resistant to Acridines and Mitoxantrone: Analysis of Cytotoxicity and Cleavable Complex Formation

Murine transgenic cell lines lacking DNA topoisomerase II (topo II)b have been used to assess the importance of topo IIb as a drug target. Western blot analysis confirmed that the topo IIb 2/2 cell lines did not contain topo IIb protein. In addition, both the topo IIb 1/1 and topo IIb 2/2 cell lines contained similar levels of topo IIa protein. The trapped in agarose DNA immunostaining assay (TARDIS) was used to detect topo IIa and b cleavable complexes in topo IIb 2/2 and topo IIb 1/1 cells. These results show that both topo IIa and b are in vivo targets for etoposide, mitoxantrone, and amsacrine (mAMSA) in topo IIb 1/1 cells. As expected, only the a-isoform was targeted in topo IIb 2/2 cells. Clonogenic assays comparing the survival of topo IIb 2/2 and topo IIb 1/1 cells were carried out to establish whether the absence of topo IIb caused drug resistance. Increased survival of topo IIb 2/2 cells compared with topo IIb 1/1 cells was observed after treatment with amsacrine (mAMSA), methyl N-(49-[9-acridinylamino]-2-methoxyphenyl) carbamate hydrochloride (AMCA), methyl N-(49-[9-acridinylamino]-2-methoxyphenyl)carbamate hydrochloride (mAMCA), mitoxantrone, and etoposide. These studies showed that topo IIb 2/2 cells were significantly more resistant to mAMSA, AMCA, mAMCA, and mitoxantrone, than topo IIb 1/1 cells, indicating that topo IIb is an important target for the cytotoxic effects of these compounds. Eukaryotic topoisomerase II (topo II) is an ATP-dependent nuclear enzyme that catalyzes changes in DNA topology. The reaction mechanism involves the passage of one DNA duplex through another by transiently cleaving a single DNA helix to create a DNA gate. During the cleavage reaction, a covalent enzyme-DNA intermediate is formed between a tyrosine residue of each topo II monomer and the 59-phosphate group of the cleaved DNA. This covalent intermediate is known as a “cleavable complex” (Wang, 1996; Austin and Marsh, 1998). Topo II has been implicated in a number of cellular processes, including DNA replication, recombination, and chromatin organization (Earnshaw et al., 1985; Chen et al., 1996; Wang, 1996). In addition, it is an important cellular target for a number of currently available antineoplastic agents (Zwelling et al., 1987; Osheroff, 1989). Mammals possess two isoforms of topo II, termed a (170 kDa) and b (180 kDa), which have been mapped to human chromosomes 17q21–22 and 3p24, respectively (Tan et al., 1992). They are highly homologous in the N-terminal threequarters (78% identity) but show lower homology in the Cterminal quarter (34% identity) (Austin et al., 1993). Topo IIa and b are differentially regulated during the cell cycle and are thought to carry out distinct cellular functions (Austin and Marsh, 1998, and references therein). Levels of topo IIa increase significantly during the S and G2/M phases of the cell cycle and subsequently decrease after mitosis (Heck et al., 1988; Prosperi et al., 1994). Topo IIa is therefore considered to be a specific marker for cellular proliferation, being required for chromosome condensation and segregation of intertwined daughter chromosomes (Heck and Earnshaw, 1986; Wang, 1996). In contrast, levels of topo IIb fluctuate less throughout the cell cycle; however, they are still cell cycle dependent (Prosperi et al., 1994; Meyer et al., 1997). The cellular function of topo IIb is as yet unknown. F. Errington is supported by a Gordon Piller Studentship from the Leukemia Research Fund (Grant 9683); Dr. E. Willmore is supported by the Leukemia Research Fund (Grant 9743); and Dr. M. J. Tilby is supported by the North of England Children’s Cancer Research Fund. ABBREVIATIONS: topo, topoisomerase; mAMSA, amsacrine; AMCA, methyl N-(49-[9-acridinylamino]-phenyl)carbamate hydrochloride; mAMCA, methyl N-(49-[9-acridinylamino]-2-methoxyphenyl)carbamate hydrochloride; FITC, fluorescein isothiocyanate; TARDIS, trapped in agarose DNA immunostaining. 0026-895X/99/061309-08$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 56:1309–1316 (1999). 1309 at A PE T Jornals on O cber 8, 2017 m oharm .aspeurnals.org D ow nladed from Topo II is an important cellular target for a number of currently available chemotherapy agents (Zwelling et al., 1987; Osheroff, 1989). These drugs inhibit the normal cellular function of topo II by stabilizing the usually transient “cleavable complex” that is formed during the catalytic cycle of the enzyme. Stabilized cleavable complexes are the primary lesions produced within the cell that initiate cell death. These are thought to be converted to permanent DNA breaks during DNA replication, causing various chromosomal aberrations (Pommier et al., 1985a; Chen et al., 1996; Suzuki et al., 1997). Studies of drug-resistant cell lines have revealed a number of factors responsible for cellular resistance to topo II inhibitors. Classical multidrug resistance, caused by overexpression of genes encoding multidrug resistance protein 1 and multidrug resistance-associated protein leads to increased efflux of exogenous toxins, including topo II inhibitors, from the cell, thereby reducing their cytotoxicity (Long et al., 1991; Lorico et al., 1995). In addition, reduced levels of topo II and genetic mutations have been identified in various drug-resistant cell lines. Further investigations of sensitive and resistant cell lines have indicated that topo IIa and b may be differentially targeted by topo II agents (Mirski et al., 1993; Dereuddre et al., 1995). Levels of topo IIa and b in neoplastic tissues can vary between tumor types (Giaccone et al., 1995). Cycling tumors contain higher levels of topo IIa than tumors with a low percentage of cycling cells, and slow-growing tumors contain significant levels of topo IIb. It also has been reported that levels of topo IIb are increased in human tumors compared with normal tissues (Turley et al., 1997). Furthermore, lymphomas and breast cancers have been shown to contain predominately more topo IIa than b, whereas seminomas contain equal levels of each isoform (Holden et al., 1992). Therefore, levels of topo IIa and b in cancerous tissues and the specificity of topo II inhibitors are important factors to consider when trying to make regimes for cancer chemotherapy more selective. This paper investigates whether topo IIa and b are differentially targeted in vivo by several topo II inhibitors. Murine cell lines totally lacking topo IIb have been used to assess the importance of topo IIa and b in the cytotoxic effects of etoposide, mitoxantrone, and three acridine derivatives, including amsacrine (mAMSA). Materials and Methods Construction of Murine Cell Lines Targeted replacement of two exons of the murine TOP2b gene, one of which contains the codon for the active site tyrosyl residue of the enzyme, with a neomycin-resistance marker was carried out following standard protocols (W.L. and J.C. Wang, unpublished data). Heterozygous TOP2b 1/2 mice were intercrossed and stage 13.5 embryos genotyped. A TOP2b 1/1 and a top2b 2/2 embryo were selected and used in the preparation of fibroblasts. Construction of immortalized fibroblasts, with simian virus 40 transformation, will be described elsewhere (L.L., W.L., and G.L., unpublished data).