[CANCER RESEARCH 60, 5937–5940, November 1, 2000] Advances in Brief Chloroquinoxaline Sulfonamide (NSC 339004) Is a Topoisomerase II␣/␤ Poison1 Hanlin Gao, Edith F. Yamasaki, Kenneth K. Chan, Linus L. Shen, and Robert M. Snapka2 Departments of Radiology [H. G., E. F. Y., R. M. S.]; Molecular Virology, Immunology and Medical Genetics [H. G., R. M. S.]; College of Medicine [H. G., E. F. Y., R. M. S., K. K. C.]; and College of Pharmacy [K. K. C.], Ohio State University, Columbus, Ohio 43210, and Abbott Laboratories, Abbott Park, Illinois 60064 [L. L. S.] Abstract Drugs and Enzymes. CQS (NSC 339004) was provided by Dr. R. Shoe- maker, National Cancer Institute. VM-26 (teniposide, NSC 122819) was Chloroquinoxaline sulfonamide (chlorosulfaquinoxaline, CQS, NSC obtained from the National Cancer Institute Division of Cancer Treatment, 339004) is active against murine and human solid tumors. On the basis of Natural Products Branch. DMSO was the solvent for all drug stocks. Purified ␤ its structural similarity to the topoisomerase II -specific drug XK469, human topoisomerase II␣ was from TopoGen (Columbus, OH) and LLS ␣ CQS was tested and found to be both a topoisomerase-II and a topoi- (Abbott Laboratories, Abbott Park, IL). Purified topoisomerase II␤ was a ␤ somerase-II poison. Topoisomerase II poisoning by CQS is essentially gift of Dr. Caroline Austin (University of Newcastle, Newcastle upon Tyne, undetectable in assays using the common protein denaturant SDS, but United Kingdom). easily detectable with strong chaotropic protein denaturants. The finding Filter Assay for in Vitro Topoisomerase-DNA Cross-links. The GF/C that detection of topoisomerase poisoning can be so dependent on the filter assay for protein-SV40 DNA cross-links is used to measure topoisomer- protein denaturant used in the assay has implications for drug discovery ase poisoning in vitro with purified enzymes and DNA substrates (9). SV40- efforts and for our understanding of topoisomerase poisons. infected cells were labeled with [3H]dThd (Amersham Pharmacia Biotech, Introduction Piscataway, NJ) at 36 h postinfection (100 ␮Ci/ml, 2 h). Labeled SV40 DNA was isolated using a Midi Plasmid isolation kit (QIAGEN, Valencia, CA). 3 CQS is a structural analogue of sulfaquinoxaline, a compound DNA (12,000 dpm) was equilibrated with or without drugs in 10 mM Tris-HCl, ␮ used to control coccidiosis in poultry, rabbit, sheep, and cattle (Fig. 1). 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 g/ml BSA and 1 mM ATP for CQS was selected for clinical development based on good activity 5 min at 37°C. The reactions were started by addition of the topoisomerase II␣ against human tumor cells in the human tumor colony-forming assay or topoisomerase II␤ and were incubated 30 min at 37°C. Various amounts of (1) and subsequently has shown activity against murine and human CQS were included in separate reactions, keeping the solvent volume constant. solid tumors (1, 2). Although CQS has been under study for over a Reactions were stopped by adding SDS (1% final concentration), GuHCl (0.4 decade and is completing Phase I trial (2) and currently moving into M final concentration), or urea (0.8 M final concentration). These protein denaturants inactivate topoisomerases trapped in topoisomerase-DNA cleav- Phase II trial, its mechanism has not been determined (3, 4). Sulfa- age complexes by topoisomerase poisons and thus render the covalent topoi- quinoxalines have been reported to possess antifolate activity (5), but somerase-DNA cross-links irreversible. To assay protein cross-links to SV40 antifolate activity has been ruled out for CQS (6, 7). CQS was also DNA, duplicate aliquots of the reaction were mixed with 0.4 M GuHCl buffer found not to intercalate into DNA (6). CQS bears a gross structural [0.4 M GuHCl, 10 mM Tris-HCl, (pH 8.0), 10 mM NaEDTA, 0.01% sarkosyl, resemblance to another solid-tumor-specific agent, XK469 (NSC and 0.3 M NaCl] and 4.0 M GuHCl, respectively, and then filtered through 697889), in that both possess chloroquinoxaline rings attached to a prewetted GF/C glass fiber filters (Whatman, Clifton, NJ; Ref. 9). In 4.0 M small aromatic ring with an acidic function (Fig. 1). XK469, an GuHCl (DNA-binding conditions), all nucleic acids bind to the filter. The herbicide analogue, is in the late stage of preclinical development. radioactivity retained on the filter under DNA binding conditions gives the Similar to CQS, several common mechanisms of biological activity value for total labeled DNA in the aliquot. In 0.4 M GuHCl buffer (protein- had been ruled out for XK469, including antimetabolite activity, DNA binding conditions), the labeled DNA retained on the filter is DNA cross- and tubulin binding, alkylation, and protein kinase inhibition (8). linked to the topoisomerase. The ratio of the radioactivity retained on GF/C Because we have recently found that XK469 is a selective topoi- filters in 0.4 M GuHCl buffer to the radioactivity retained on filters in 4.0 M somerase II␤ poison (9), we tested CQS for inhibition of topoisomer- GuHCl gives the fraction of labeled DNA that is cross-linked to the topoi- ases and found it to be both a topoisomerase II␣ and topoisomerase somerase. A single covalently cross-linked protein is sufficient to cause the retention of a DNA molecule as large as the adenovirus genome (35,937 bp) II␤ poison. Detection of topoisomerase poisoning by CQS requires on the filter under protein-binding conditions (10). In the absence of added strong chaotropic protein denaturants, such as GuHCl or urea, rather topoisomerase or drugs (reaction buffer with [3H]dThd-labeled SV40 DNA), than the more commonly used detergent, SDS. approximately 1–2% of the substrate DNA is retained on the filters in 0.4-M GuHCl buffer (protein-binding conditions). Because as there is some variabil- Materials and Methods ity in the specific activity of topoisomerase preparations, the assay is adjusted Cells. African green monkey cells (CV-1) were obtained from the Ameri- for each batch of topoisomerase. Sufficient topoisomerase II is added to the can Type Culture Collection and were maintained in Eagle’s MEM (Life reaction for ϳ2–3% SDS-induced topoisomerase-DNA cross-linking in the Technologies, Inc., Grand Island, NY) supplemented with 10% calf serum, 14 presence of the drug solvent (DMSO) alone. This concentration of topoisomer- ase thus results in steady-state levels of topoisomerase-DNA cleavage com- mM Hepes (pH 7.2), 4 mM NaHCO3, and penicillin/streptomycin. plexes sufficient for detection in the absence of topoisomerase poisons. A value of 4–5% cross-linking in the absence of added topoisomerase poisons is Received 5/22/00; accepted 9/13/00. The costs of publication of this article were defrayed in part by the payment of page thus attributable to 1–2% nonspecific DNA binding to the filter and 2–3% charges. This article must therefore be hereby marked advertisement in accordance with background topoisomerase II-DNA cleavage complexes. Drug-induced topoi- 18 U.S.C. Section 1734 solely to indicate this fact. somerase-DNA cross-links above this value are taken as a measure of topoi- 1 Supported by grants from the Public Health Service, NCI RO1 CA80961 to R. M. S., Contract NO1-CM-57201 to K. K. C., U01CA63185 to K. K. C. and R. M. S., and P30 somerase poisoning. Each drug studied is also tested in reaction buffer without CA16058 to The Ohio State University Comprehensive Cancer Center. topoisomerase to ensure that it does not cause DNA binding to the GF/C filter 2 To whom requests for reprints should be addressed, at Ohio State University, in 0.4 M GuHCl buffer. When GuHCl is used to stop the topoisomerase th Department of Radiology, 103 Wiseman Hall, 400 West 12 Avenue, Columbus, OH reaction, the topoisomerase-DNA cross-linking value for the “solvent only” 43210. Phone: (614) 292-9375; Fax: (614) 292-7237. 3 The abbreviations used are: CQS, chloroquinoxaline sulfonamide; GuHCl, guani- (i.e., no drug) control is always slightly higher than it is for an identical dinium chloride; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. reaction stopped by the addition of SDS. This may be attributable to more rapid 5937 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. TOPOISOMERASE POISONING BY CQS For assay of topoisomerase II␣-dependent DNA cleavage, reactions con- tained end-labeled DNA fragments (50,000 dpm/reaction), 10 mM Hepes-HCl (pH 7.9), 50 mM KCl, 5 mM MgCl2,50mM NaCl, 0.1 mM Na2EDTA, and 1 mM ATP. After a 5-min preincubation at 37°C, the reaction was started by addition of 1.2 ␮g of purified human topoisomerase II␣ (total reaction volume, 20 ␮l). The reaction mix was incubated at 37°C for 30 min before being terminated by the addition of 2 ␮lof4M GuHCl. The DNA was purified by ethanol precipitation, then resuspended in 28 ␮l of proteinase K solution (0.2 mg/ml, 2 h, 45°C). The DNA was repurified by ethanol precipitation before resuspension in 4 ␮l of loading buffer (80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue). Samples were heated to 95°C for 5 min, cooled to room temperature, and then loaded onto a DNA sequencing gel (8% polyacrylamide, 19:1 acrylamide/bisacrylamide) contain- ing 7 M urea in 1 ϫ Tris-borate/EDTA buffer (11). Electrophoresis was performed at 1,400 V for 1.5 h. The gel was transferred to Whatman No. 3 MM paper and exposed to Hyperfilm-MP (Amersham Pharmacia Biotech). Cytotoxicity Assay. The MTT reduction assay (12, 13)was used to deter- mine the cytotoxicity of CQS for CV-1 cells.