cancer res. 2000; 60, 5937-5940 chloroquinoxaline sulfonamide (nsc 339004) is a topoisomerase iiab...

8/6/2019 Cancer Res. 2000; 60, 5937-5940 Chloroquinoxaline Sulfonamide (NSC 339004) is a Topoisomerase IIab Poison

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2000;60:5937-5940. Published online November 1, 2000.Cancer ResHanlin Gao, Edith F. Yamasaki, Kenneth K. Chan, et al.

Poison/Topoisomerase IIChloroquinoxaline Sulfonamide (NSC 339004) Is a

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[CANCER RESEARCH 60, 59375940, 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. Snapka 2

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

Chloroquinoxaline sulfonamide (chlorosulfaquinoxaline, CQS, NSC

339004) is active against murine and human solid tumors. On the basis of

its structural similarity to the topoisomerase II-specific drug XK469,

CQS was tested and found to be both a topoisomerase-II and a topoi-

somerase-II poison. Topoisomerase II poisoning by CQS is essentially

undetectable in assays using the common protein denaturant SDS, but

easily detectable with strong chaotropic protein denaturants. The finding

that detection of topoisomerase poisoning can be so dependent on the

protein denaturant used in the assay has implications for drug discovery

efforts and for our understanding of topoisomerase poisons.

Introduction

CQS

3

is a structural analogue of sulfaquinoxaline, a compoundused to control coccidiosis in poultry, rabbit, sheep, and cattle (Fig. 1).

CQS was selected for clinical development based on good activity

against human tumor cells in the human tumor colony-forming assay

(1) and subsequently has shown activity against murine and human

solid tumors (1, 2). Although CQS has been under study for over a

decade and is completing Phase I trial (2) and currently moving into

Phase II trial, its mechanism has not been determined (3, 4). Sulfa-

quinoxalines have been reported to possess antifolate activity (5), but

antifolate activity has been ruled out for CQS (6, 7). CQS was also

found not to intercalate into DNA (6). CQS bears a gross structural

resemblance to another solid-tumor-specific agent, XK469 (NSC

697889), in that both possess chloroquinoxaline rings attached to a

small aromatic ring with an acidic function (Fig. 1). XK469, an

herbicide analogue, is in the late stage of preclinical development.Similar to CQS, several common mechanisms of biological activity

had been ruled out for XK469, including antimetabolite activity, DNA

and tubulin binding, alkylation, and protein kinase inhibition (8).

Because we have recently found that XK469 is a selective topoi-

somerase II poison (9), we tested CQS for inhibition of topoisomer-

ases and found it to be both a topoisomerase II and topoisomerase

II poison. Detection of topoisomerase poisoning by CQS requires

strong chaotropic protein denaturants, such as GuHCl or urea, rather

than the more commonly used detergent, SDS.

Materials and Methods

Cells. African green monkey cells (CV-1) were obtained from the Ameri-

can Type Culture Collection and were maintained in Eagles MEM (Life

Technologies, Inc., Grand Island, NY) supplemented with 10% calf serum, 14

mM Hepes (pH 7.2), 4 mM NaHCO3, and penicillin/streptomycin.

Drugs and Enzymes. CQS (NSC 339004) was provided by Dr. R. Shoe-

maker, National Cancer Institute. VM-26 (teniposide, NSC 122819) was

obtained from the National Cancer Institute Division of Cancer Treatment,

Natural Products Branch. DMSO was the solvent for all drug stocks. Purified

human topoisomerase II was from TopoGen (Columbus, OH) and LLS

(Abbott Laboratories, Abbott Park, IL). Purified topoisomerase II was a

gift of Dr. Caroline Austin (University of Newcastle, Newcastle upon Tyne,

United Kingdom).

Filter Assay for in Vitro Topoisomerase-DNA Cross-links. The GF/C

filter assay for protein-SV40 DNA cross-links is used to measure topoisomer-

ase poisoning in vitro with purified enzymes and DNA substrates (9). SV40-

infected cells were labeled with [3H]dThd (Amersham Pharmacia Biotech,

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).

DNA (12,000 dpm) was equilibrated with or without drugs in 10 m M Tris-HCl,50 mM KCl, 5 mM MgCl

2, 0.1 mM EDTA, 15 g/ml BSA and 1 mM ATP for

5 min at 37C. The reactions were started by addition of the topoisomerase II

or topoisomerase II and were incubated 30 min at 37C. Various amounts of

CQS were included in separate reactions, keeping the solvent volume constant.

Reactions were stopped by adding SDS (1% final concentration), GuHCl (0.4

M final concentration), or urea (0.8 M final concentration). These protein

denaturants inactivate topoisomerases trapped in topoisomerase-DNA cleav-

age complexes by topoisomerase poisons and thus render the covalent topoi-

somerase-DNA cross-links irreversible. To assay protein cross-links to SV40

DNA, duplicate aliquots of the reaction were mixed with 0.4 M GuHCl buffer

[0.4 M GuHCl, 10 mM Tris-HCl, (pH 8.0), 10 mM NaEDTA, 0.01% sarkosyl,

and 0.3 M NaCl] and 4.0 M GuHCl, respectively, and then filtered through

prewetted GF/C glass fiber filters (Whatman, Clifton, NJ; Ref. 9). In 4.0 M

GuHCl (DNA-binding conditions), all nucleic acids bind to the filter. The

radioactivity retained on the filter under DNA binding conditions gives thevalue for total labeled DNA in the aliquot. In 0.4 M GuHCl buffer (protein-

binding conditions), the labeled DNA retained on the filter is DNA cross-

linked to the topoisomerase. The ratio of the radioactivity retained on GF/C

filters in 0.4 M GuHCl buffer to the radioactivity retained on filters in 4.0 M

GuHCl gives the fraction of labeled DNA that is cross-linked to the topoi-

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)

on the filter under protein-binding conditions (10). In the absence of added

topoisomerase or drugs (reaction buffer with [3H]dThd-labeled SV40 DNA),

approximately 12% of the substrate DNA is retained on the filters in 0.4-M

GuHCl buffer (protein-binding conditions). Because as there is some variabil-

ity in the specific activity of topoisomerase preparations, the assay is adjusted

for each batch of topoisomerase. Sufficient topoisomerase II is added to the

reaction for 23% SDS-induced topoisomerase-DNA cross-linking in the

presence of the drug solvent (DMSO) alone. This concentration of topoisomer-

ase thus results in steady-state levels of topoisomerase-DNA cleavage com-

plexes sufficient for detection in the absence of topoisomerase poisons. A

value of 45% cross-linking in the absence of added topoisomerase poisons is

thus attributable to 12% nonspecific DNA binding to the filter and 23%

background topoisomerase II-DNA cleavage complexes. Drug-induced topoi-

somerase-DNA cross-links above this value are taken as a measure of topoi-

somerase poisoning. Each drug studied is also tested in reaction buffer without

topoisomerase to ensure that it does not cause DNA binding to the GF/C filter

in 0.4 M GuHCl buffer. When GuHCl is used to stop the topoisomerase

reaction, the topoisomerase-DNA cross-linking value for the solvent only

(i.e., no drug) control is always slightly higher than it is for an identical

reaction stopped by the addition of SDS. This may be attributable to more rapid

Received 5/22/00; accepted 9/13/00.

The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with

18 U.S.C. Section 1734 solely to indicate this fact.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

CA16058 to The Ohio State University Comprehensive Cancer Center.2 To whom requests for reprints should be addressed, at Ohio State University,

Department of Radiology, 103 Wiseman Hall, 400 West 12th Avenue, Columbus, OH

43210. Phone: (614) 292-9375; Fax: (614) 292-7237.3 The abbreviations used are: CQS, chloroquinoxaline sulfonamide; GuHCl, guani-

dinium chloride; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.

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protein denaturation by the chaotropic denaturant GuHCl, resulting in more

efficient trapping of topoisomerase-DNA cleavage complexes.

Filter Assay for Cellular Protein-DNA Cross-links. CV-1 cells in earlyconfluence were labeled with [3H]dThd (1.0 Ci/ml, 43 h) by adding label

directly to the medium. Drug treatments were carried out for 15 min on the

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

containing a small stainless steel nut, the tube capped securely, and the DNA

sheared by vortexing for 15 s. The lysate then was heated at 65C for 10 min

to ensure denaturation and removal of noncovalently attached proteins from

the DNA. After cooling to room temperature, aliquots of the lysate were

assayed with the GF/C filter assay for the percentage of labeled DNA that is

cross-linked to protein as in the assay for protein-SV40 DNA cross-links. As

in the in vitro assay for topoisomerase-DNA cross-links (above), GF/C glass

fiber filter binding in 4 M GuHCl gives a value for the total radiolabeled DNA

in the aliquot, and the filter-binding in 0.4 M GuHCl buffer gives a value for

protein-DNA cross-links. A variation of this assay, in which SDS is used to

lyse the cells and render topoisomerase-DNA cleavage complexes irreversible,has been described (9). In the SDS-lysis-based assay, the level of protein-DNA

cross-linking in the absence of added topoisomerase poisons is typically

510%. Proteinase K digestion of such lysates reduces the level of cross-

linking to 12%. This suggests that a 510% value for protein-DNA cross-

linking in the absence of added topoisomerase poisons represents 12%

because of nonspecific DNA binding to filters (similar to the in vitro assay

described above) and 38% 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 topoisomer-

ase-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 topoi-

somerase isozyme.

Topoisomerase II

-Induced DNA Cleavage Reaction.A 516-bp DNAsubstrate (residues 38464362 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 32P in a 40-l reaction

containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 50 g/ml

acetylated BSA, 0.25 mM of each deoxynucleotide (dGTP, dCTP, dTTP), and

70 Ci [-32P]dATP (800 Ci/mmol) and Klenow fragment (5 units, USB Corp.

Cleveland, OH). After a 15-min incubation at 37C, unlabeled dCTP, dGTP,

dTTP, and dATP were added (10 nM of each), and the incubation was

continued for an additional 15 min before termination by heating at 70C for

10 min. The end-labeled DNA fragment was then purified with a mini-Quick

Spin DNA column (Roche, Indianapolis, IN).

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, 50 mM NaCl, 0.1 mM Na2EDTA, and 1

mM ATP. After a 5-min preincubation at 37C, 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 37C for 30 min before being

terminated by the addition of 2 l of 4 M GuHCl. The DNA was purified by

ethanol precipitation, then resuspended in 28 l of proteinase K solution (0.2

mg/ml, 2 h, 45C). 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 95C 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 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 2.5 104 cells/well in 96-well tissue culture

plates, and then incubated at 37C 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

0.5 mg/ml and the incubation was continued for 5 h at 37C. The medium was

then replaced with 100% N,N-dimethylformamide (100 l/well), and the plates

were left at 37C for another 2 h. Then, colorimetric analysis at 550 nm was

done. Values in the presence of the drug solvent alone were used as the blank

control.

Results and Discussion

CQS caused dose-dependent protein-DNA cross-links to CV-1

monkey kidney cell chromosomal DNA when drug treatment was

terminated by lysis with GuHCl (Fig. 2). The m M concentration range

is achievable clinically. In an early Phase I clinical trial at an i.v. dose

of 4060 mg/m2 every 28 days, peak plasma concentrations of higher

than 1 mM (500 g/ml) was achieved (14). In a subsequent Phase I

clinical trial using a 2000-mg/m2 dose weekly for 4 weeks, plasma

concentration at0.3 mM (or 100 g/ml) concentrations was found

(2). The CQS IC50

for CV-1cells, obtained using an MTT cytotoxicity

assay, was 1.8 mM (data not shown). CQS lacks functional groups thatwould make it a bifunctional protein-DNA cross-linking agent, and

the short drug exposure (15 min) allows little time for metabolism.

When the same assay was done using SDS for cell lysis, no CQS-

Fig. 1. Structures of sulfaquinoxaline, CQS, and XK469.

Fig. 2. CQS-induced protein-DNA cross-links in CV-1 cells. CV-1 monkey kidney

cells in early confluence were labeled with [3H]dThd for 43 h by adding the label directly

to the medium. The cells were treated with CQS for 15 min. The medium and drug were

removed and the cells lysed with 6 M GuHCl. The lysate was vortexed as described (9) to

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 theabsence of CQS, has been subtracted from each measurement (see Materials and

Methods).

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TOPOISOMERASE POISONING BY CQS



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induced protein-DNA cross-links were detected. Because dose-depen-

dent protein-DNA cross-linking is also characteristic of topoisomer-

ase 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 physio-

logical 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 topoi-

somerase 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 detecttopoisomerase 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-spe-

cific differences in binding are implied. This, in turn, predicts that

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 co-

valently 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 irre-

versible protein-associated DNA strand break by the protein dena-

turant. However, enzymatic inactivation of the topoisomerase by

complete denaturation may not be an instantaneous process. Complete

denaturation is likely to require interaction with a number of dena-

turant molecules. We propose that the binding of the first few mole-

cules of SDS may alter the structure of CQS-stabilized topoisomerase

II-DNA cleavage complexes so that they release the CQS molecule

while retaining enough structure to carry out the religation step of the

topoisomerase reaction. Denaturation caused by a stronger protein

Fig. 3. CQS-induced topoisomerase II-DNA cross-links. A, purified [3H]dThd-labeledSV40 DNA was incubated with purified topoisomerase II (TopoGen batch AP 159) or

topoisomerase II in the presence of CQS at the concentrations indicated. The reactions

were stopped by the addition of GuHCl (E, topoisomerase II; , topoisomerase II) or

SDS (F, topoisomerase II; f, topoisomerase II) and assayed for topoisomerase-DNA

cross-links. B, [3H]dThd-labeled SV40 DNA was incubated with purified human topoi-

somerase II (TopoGen batch FB 1400) either with CQS (1 g/ml, white bars) or withoutCQS (black bars); the reactions were stopped with the indicated protein denaturants and

assayed for topoisomerase-DNA cross-links.

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 topoisomer-

ase II with 3.3 mM CQS (37C, 30 min). The reactions were terminated by the addition

of GuHCl. DNA was purified from each sample, denatured by heating at 95C in 80%

formamide, 10 mM

NaOH, 1 mM

EDTA, cooled, and loaded on a DNA sequencing gel forelectrophoretic 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).

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denaturant may inactivate the topoisomerases in CQS-stabilized DNA

cleavage intermediates so rapidly that they cannot complete their

reactions.

CQS and XK469 are both quinoxalines. Although there are signif-

icant differences in structure, there are also strong similarities that led

us to test the topoisomerase activity of CQS. Both compounds share

a quinoxaline ring that is linked to a parasubstituted phenyl ring with

a bridge at the 2 position of the quinoxaline ring. These two com-

pounds also possess acidic moieties. In CQS, the acidic sulfonamide

function is located in the linker between the two ring systems, whereas

the acidic propionic acid function of XK469 is exo to the ring system.

Both molecules can adopt conformations that place the acidic function

near the quinoxaline ring. CQS and XK469 also differ in the phenyl

ring system, with CQS having a basic amino group that is absent in

XK469.

XK469 and CQS represent the first members of a new quinoxaline

class of topoisomerase II inhibitors. Because both drugs show solid

tumor activity, this may be a general characteristic of the quinoxaline

topoisomerase II poisons. Both drugs are very weak topoisomerase II

poisons with low nonspecific cytotoxicity, so high therapeutic doses

can be tolerated. Although XK469 is very selective for the isozyme

of topoisomerase II (p180), CQS appears to target both the and the

(p170) isozymes. The basis of isozyme selectivity for these drugs is

not readily apparent, but it may be related to the differences infunctionalities and/or regio-alignment with the quinoxaline ring. Ad-

ditional insights into topoisomerase II isozyme selectivity may be

accomplished through structure-activity studies.

Acknowledgments

We thank TopoGen (Columbus, Ohio) for purified human topoisomerase

II and Dr. Caroline Austin (University of Newcastle, United Kingdom) for

purified human topoisomerase II.

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