Chloroquinoxaline Sulfonamide (NSC 339004) Is a Topoisomerase II␣/␤ Poison1

[CANCER RESEARCH 60, 5937–5940, November 1, 2000] Advances in Brief

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 topoisomerase 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

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) containing 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. In this assay, a tetrazolium salt, MTT, was used as a colorimetric substrate for measurements of cell viability. Cells were plated at a density of 2.5 ϫ 104 cells/well in 96-well tissue culture Fig. 1. Structures of sulfaquinoxaline, CQS, and XK469. plates, and then incubated at 37°C in MEM medium with 10% FCS. After 24 h incubation, different concentrations of drug were added, and incubation was continued for another 3 days. MTT was then added to a final concentration of protein denaturation by the chaotropic denaturant GuHCl, resulting in more 0.5 mg/ml and the incubation was continued for5hat37°C. The medium was efficient trapping of topoisomerase-DNA cleavage complexes. then replaced with 100% N,N-dimethylformamide (100 ␮l/well), and the plates Filter Assay for Cellular Protein-DNA Cross-links. CV-1 cells in early were left at 37°C for another 2 h. Then, colorimetric analysis at 550 nm was confluence were labeled with [3H]dThd (1.0 ␮Ci/ml, 43 h) by adding label done. Values in the presence of the drug solvent alone were used as the blank directly to the medium. Drug treatments were carried out for 15 min on the control. cells. Then the medium was removed, and the cells were lysed with 6 M GuHCl. The lysate (500 ␮l) was transferred to a 1.5-ml microcentrifuge tube Results and Discussion containing a small stainless steel nut, the tube capped securely, and the DNA CQS caused dose-dependent protein-DNA cross-links to CV-1 sheared by vortexing for 15 s. The lysate then was heated at 65°C for 10 min monkey kidney cell chromosomal DNA when drug treatment was to ensure denaturation and removal of noncovalently attached proteins from the DNA. After cooling to room temperature, aliquots of the lysate were terminated by lysis with GuHCl (Fig. 2). The mM concentration range assayed with the GF/C filter assay for the percentage of labeled DNA that is is achievable clinically. In an early Phase I clinical trial at an i.v. dose 2 cross-linked to protein as in the assay for protein-SV40 DNA cross-links. As of 4060 mg/m every 28 days, peak plasma concentrations of higher in the in vitro assay for topoisomerase-DNA cross-links (above), GF/C glass than 1 mM (Ͼ500 ␮g/ml) was achieved (14). In a subsequent Phase I 2 fiber filter binding in 4 M GuHCl gives a value for the total radiolabeled DNA clinical trial using a 2000-mg/m dose weekly for 4 weeks, plasma in the aliquot, and the filter-binding in 0.4 M GuHCl buffer gives a value for concentration at Ͼ0.3 mM (or Ͼ100 ␮g/ml) concentrations was found protein-DNA cross-links. A variation of this assay, in which SDS is used to (2). The CQS IC50 for CV-1cells, obtained using an MTT cytotoxicity lyse the cells and render topoisomerase-DNA cleavage complexes irreversible, assay, was 1.8 mM (data not shown). CQS lacks functional groups that has been described (9). In the SDS-lysis-based assay, the level of protein-DNA would make it a bifunctional protein-DNA cross-linking agent, and cross-linking in the absence of added topoisomerase poisons is typically the short drug exposure (15 min) allows little time for metabolism. ϳ5–10%. Proteinase K digestion of such lysates reduces the level of cross- linking to ϳ1–2%. This suggests that a 5–10% value for protein-DNA cross- When the same assay was done using SDS for cell lysis, no CQS- linking in the absence of added topoisomerase poisons represents 1–2% because of nonspecific DNA binding to filters (similar to the in vitro assay described above) and 3–8% because of trapping of endogenous topoisomerase- DNA cleavage complexes. In contrast to the in vitro assay, where a single purified topoisomerase is added to the reaction mix, the background protein- DNA cross-linking value in cells is assumed to represent trapped topoisomerase-DNA cleavage complexes of a number of different type-I and type-II topoisomerases active in the intact cells. Thus, topoisomerase poisoning meas- ured in this in vivo assay may represent poisoning of more than one topoisomerase isozyme. Topoisomerase II␣-Induced DNA Cleavage Reaction. A 516-bp DNA substrate (residues 3846–4362 in pBR322) was labeled on one end as follows: pBR322 plasmid DNA was digested with EcoRI and ScaI to generate a fragment with one blunt end and one sticky end. The DNA fragment was purified by agarose gel electrophoresis, band excision, and a Gel Extraction kit (QIAGEN). The overhang end was labeled with 32Pina40-␮l reaction ␮ containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2,1mM DTT, 50 g/ml acetylated BSA, 0.25 mM of each deoxynucleotide (dGTP, dCTP, dTTP), and Fig. 2. CQS-induced protein-DNA cross-links in CV-1 cells. CV-1 monkey kidney ␮ ␣ 32 70 Ci [ - P]dATP (800 Ci/mmol) and Klenow fragment (5 units, USB Corp. cells in early confluence were labeled with [3H]dThd for 43 h by adding the label directly Cleveland, OH). After a 15-min incubation at 37°C, unlabeled dCTP, dGTP, to the medium. The cells were treated with CQS for 15 min. The medium and drug were dTTP, and dATP were added (10 nM of each), and the incubation was removed and the cells lysed with 6 M GuHCl. The lysate was vortexed as described (9) to continued for an additional 15 min before termination by heating at 70°C for reduce the DNA size by shearing. Aliquots of the cell lysate were then assayed for protein DNA cross-links using the GF/C filter assay. A 7% background binding, seen in the 10 min. The end-labeled DNA fragment was then purified with a mini-Quick absence of CQS, has been subtracted from each measurement (see “Materials and Spin DNA column (Roche, Indianapolis, IN). Methods”). 5938

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. TOPOISOMERASE POISONING BY CQS induced protein-DNA cross-links were detected. Because dose-dependent protein-DNA cross-linking is also characteristic of topoisomerase poisons, we tested CQS against purified topoisomerase II␣ and II␤ in an in vitro assay for topoisomerase poisoning. As shown in Fig. 3A, CQS caused cross-linking of both human topoisomerase II isozymes to the substrate DNA in a concentration-dependent manner when GuHCl was used to terminate the reaction but not when SDS was used to terminate the reaction. Because SDS is negatively charged and GuHCl is positively charged at physiological pH, they were compared with another protein denaturant, urea, which is uncharged at physiological pH. Urea, like GuHCl, proved to be an efficient protein denaturant for detection of topoisomerase II poisoning by CQS (Fig. 3B). For additional confirmation of topoisomerase poisoning, we tested CQS with human topoisomerase II␣ in a DNA cleavage assay using a 32P-end labeled DNA substrate. As shown in Fig. 4, CQS stabilized topoisomerase II␣ cleavages. The strong topoisomerase II␣/II␤ poison, VM-26, at a lower concentration, stabilized topoisomerase II␣ cleavages at more sites on the same substrate DNA. Topoisomerase II poisoning by XK469 is readily detectable using either the detergent SDS or the chaotropic protein denaturant GuHCl (9). In contrast, detection of topoisomerase II poisoning by CQS requires strong chaotropic protein denaturants, such as GuHCl and urea, and is essentially undetectable with SDS. The requirement of a strong protein denaturant, like GuHCl, to detect topoisomerase poisoning by CQS appears to be unique. We are not aware of any previous reports of topoisomerase poisons with this characteristic. The almost universal use of SDS in topoisomerase poisoning assays may be the reason that the topoisomerase II activity of CQS was not discovered during its many years of development as an anticancer drug. Because XK469 shows isozyme selectivity in topoisomerase II poisoning, isozyme-specific differences in binding are implied. This, in turn, predicts that

Fig. 4. Stimulation of topoisomerase II␣-DNA cleavage by CQS and VM-26. A uniquely 32P-end-labeled 516-bp restriction fragment of pBR322 was incubated with human topoisomerase II␣ alone, topoisomerase II␣ with 100 ␮M VM-26, or topoisomerase II␣ with 3.3 mM CQS (37°C, 30 min). The reactions were terminated by the addition of GuHCl. DNA was purified from each sample, denatured by heating at 95°C in 80% formamide, 10 mM NaOH, 1 mM EDTA, cooled, and loaded on a DNA sequencing gel for electrophoretic separation of cleaved DNA. Lanes marked “DNA” included the substrate DNA in identical reaction mixtures, but without topoisomerase. CQS did not cause DNA strand breaks in the absence of topoisomerase (not shown).

drugs may be found that act as poisons of both topoisomerase II isozymes but whose poisoning of one or the other isozyme requires strong chaotropic denaturants for detection. These findings also raise the possibility that extensive drug discovery efforts focused on topoisomerase poisons and using SDS as a protein denaturant may have missed many active compounds. It is thought that topoisomerase poisons stabilize DNA strand- passing reaction intermediates in which the topoisomerase is covalently attached to the DNA at the site of a DNA strand break. Topoisomerase poison assays use protein denaturants to inactivate the topoisomerase while this reaction intermediate is stabilized by the drug. The DNA strand-passing intermediate is converted to an irreversible “protein-associated DNA strand break” by the protein denaturant. However, enzymatic inactivation of the topoisomerase by Fig. 3. CQS-induced topoisomerase II-DNA cross-links. A, purified [3H]dThd-labeled complete denaturation may not be an instantaneous process. Complete SV40 DNA was incubated with purified topoisomerase II␣ (TopoGen batch AP 159) or denaturation is likely to require interaction with a number of dena- ␤ topoisomerase II in the presence of CQS at the concentrations indicated. The reactions turant molecules. We propose that the binding of the first few mole- were stopped by the addition of GuHCl (E, topoisomerase II␣; Ⅺ, topoisomerase II␤)or SDS (F, topoisomerase II␣; f, topoisomerase II␤) and assayed for topoisomerase-DNA cules of SDS may alter the structure of CQS-stabilized topoisomerase cross-links. B,[3H]dThd-labeled SV40 DNA was incubated with purified human topoi- II-DNA cleavage complexes so that they release the CQS molecule somerase II␣ (TopoGen batch FB 1400) either with CQS (1 ␮g/ml, white bars) or without CQS (black bars); the reactions were stopped with the indicated protein denaturants and while retaining enough structure to carry out the religation step of the assayed for topoisomerase-DNA cross-links. topoisomerase reaction. Denaturation caused by a stronger protein 5939

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. TOPOISOMERASE POISONING BY CQS denaturant may inactivate the topoisomerases in CQS-stabilized DNA References cleavage intermediates so rapidly that they cannot complete their 1. Shoemaker, R. H. New approaches to anticancer drug screening: the human tumor reactions. colony-forming assay. Cancer Treat. Rep., 70: 9–12, 1986. CQS and XK469 are both quinoxalines. Although there are signif- 2. Rigas, J. R., Miller, V. A., Tong, W. P., Roistacher, N., Kris, M. G., Orazem, J. P., Young, C. W., and Warrell, R. P., Jr. Clinical and pharmacology study of chloroqui- icant differences in structure, there are also strong similarities that led noxaline sulfonamide given on a weekly schedule. Cancer Chemother. Pharmacol., us to test the topoisomerase activity of CQS. Both compounds share 35: 483–488, 1995. a quinoxaline ring that is linked to a parasubstituted phenyl ring with 3. Branda, R. F., Moore, A. L., and McCormack, J. J. Immunosuppressive properties of chloroquinoxaline sulfonamide. Biochem. Pharmacol., 38: 3521–3526, 1989. a bridge at the 2 position of the quinoxaline ring. These two com- 4. Tong, W. P. Chloroquinoxaline sulfonamide. Investigational drug brochure. Be- pounds also possess acidic moieties. In CQS, the acidic sulfonamide thesda, MD: National Cancer Institute, Division of Cancer Treatment, 1987. function is located in the linker between the two ring systems, whereas 5. Poe, M. Antibacterial synergism: a proposal for chemotherapeutic potential between trimethoprim and sulfamethoxazole. Science (Washington DC), 194: 533–535, 1976. the acidic propionic acid function of XK469 is exo to the ring system. 6. Branda, R. F., McCormack, J. J., and Perlmutter, C. A. Cellular pharmacology of Both molecules can adopt conformations that place the acidic function chloroquinoxaline sulfonamide and a related compound in murine B16 melanoma near the quinoxaline ring. CQS and XK469 also differ in the phenyl cells. Biochem. Pharmacol., 37: 4557–4564, 1988. 7. Hickey, R., Schiffer, J., Wei, J., and Malkas, L. DNA synthesis is differentially ring system, with CQS having a basic amino group that is absent in affected by the drugs merbarone and chloroquinoxaline sulfonamide. Proc. Am. XK469. Assoc. Cancer Res., 34: 352, 1993. XK469 and CQS represent the first members of a new quinoxaline 8. Corbett, T. H., LoRusso, P., Demchick, L., Simpson, C., Pugh, S., White, K., Kushner, J., Polin, L., Meyer, J., Czarnecki, J., Heibrun, L., Horwitz, J. P., Gross, class of topoisomerase II inhibitors. Because both drugs show solid J. L., Behrens, C. H., Harrison, B. A., McRipley, R. J., and Trainor, G. Preclinical tumor activity, this may be a general characteristic of the quinoxaline antitumor efficacy of analogs of XK469: sodium-(2-[4-(7-chloro-2-quinoxalinyl- topoisomerase II poisons. Both drugs are very weak topoisomerase II oxy)phenoxy]propionate. Investig. New Drugs, 16: 129–139, 1998. 9. Gao, H., Huang, K. C., Yamasaki, E. F., Chan, K. K., Chohan, L., and Snapka, R. M. poisons with low nonspecific cytotoxicity, so high therapeutic doses XK469, a selective topoisomerase IIb poison. Proc. Natl. Acad. Sci. USA, 96: can be tolerated. Although XK469 is very selective for the ␤ isozyme 12168–12173, 1999. of topoisomerase II (p180), CQS appears to target both the ␤ and the 10. Coombs, D. H., and Pearson, G. D. Filter-binding assay for covalent DNA-protein ␣ complexes: adenovirus DNA terminal protein complex. Proc. Natl. Acad. Sci. USA, (p170) isozymes. The basis of isozyme selectivity for these drugs is 75: 5291–5295, 1978. not readily apparent, but it may be related to the differences in 11. Felix, C. A., Lange, B. J., Hosler, M. R., Fertala, J., and Bjornsti, M. A. Chromosome functionalities and/or regio-alignment with the quinoxaline ring. Ad- band 11q23 translocation breakpoints are DNA topoisomerase II cleavage sites. Cancer Res., 55: 4287–4292, 1995. ditional insights into topoisomerase II isozyme selectivity may be 12. Hansen, M. B., Nielsen, S. E., and Berg, K. Re-examination and further development accomplished through structure-activity studies. of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods, 119: 203–210, 1989. Acknowledgments 13. Shearman, M. S., Ragan, C. I., and Iversen, L. L. Inhibition of PC12 cell redox activity is a specific, early indicator of the mechanism of ␤-amyloid-mediated cell death. Proc. Natl. Acad. Sci. USA, 91: 1470–1474, 1994. We thank TopoGen (Columbus, Ohio) for purified human topoisomerase 14. Rigas, J. R., Tong, W. P., Kris, M. G., Orazem, J. P., Young, C. W., and Warrell, II␣ and Dr. Caroline Austin (University of Newcastle, United Kingdom) for R. P., Jr. Phase I clinical and pharmacological study of chloroquinoxaline sulfona- purified human topoisomerase II␤. mide. Cancer Res., 52: 6619–6623, 1992.

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Hanlin Gao, Edith F. Yamasaki, Kenneth K. Chan, et al.

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