topoisomerases I and II. Effect of pyrazoloacridine ...

Effect of pyrazoloacridine (NSC 366140) on DNA topoisomerases I and II. A A Adjei, M Charron, E K Rowinsky, et al. Clin Cancer Res 1998;4:683-691. Published online March 1, 1998.

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Vol.

4, 683-691,

Marc/i

Effect

/998

Clinical

of Pyrazoloacridine

Topoisomerases

Alex

A. Adjei,

Eric

K.

I and

Martin

Phyllis

Miller,

Judith

Joel

M.

Svingen,

Matthew

M.

Ames,

in intranuclear

those

required

these

results

and

(NSC

mechanism solid cells,

clinical

trials.

of action,

tumor cells, and promising In

the

(topo)

strains

functional

lacking

clear

extracts,

topo

II, and

genic

activity topo

purified

intact

assays

has shown

cytotoxicity antitumor

present

topoisomerase

selective

the

effect

was

evaluated

of

PA

using

tissue

topo

culture

cytotoxicity

on

ited

covalent

topo-DNA

topotecan-induced

complexes

and

DNA

complexes

these

results,

complexes.

I and

stabilization

colony-forming

assays

term PA exposure inhibited the and etoposide, whereas prolonged toxic to these cells. Accumulation was

concentrated

as much

topo

Received

5/8/97:

revised

1 1/6/97:

council Society 2 Present

Research

18 U.S.C.

from

the

CA73709

Institute

for Drug

is a Scholar Research.

of the

of

and

retains

In view

in several

clinical

shown

to cause cells

delayed

(10),

DNA

Hep studies,

with

PA-induced ethidium doxorubicin

DNA

(12.

solely

to UOl

fact

that

CA06973 Research

amsacrine;

on topo

Refs.

I and

topo

achievable

II

of other

(13.

other

D; Refs.

PA

nary

13)

( 13), suggesting

and

topo

from

iii

vitro

dual

accounts

PA is an intercalating

16-18),

we

II. Results

I/topo and

of

the

this

the

by

of

1301 Guggenheim.

SW.

55905.

Rochester,

MN

for

reprints

should

be

Therapy

and

San

Antonio.

TX

addressed,

Mayo

Clinic.

that

In view

topo

I (e.g.,

doxorubicin effect

topo

differs

(21).

that I and

from

as saintopin

presented

of

indicate

inhibit that

of

plasmid

the

indoloquinolinediones

that

(19), Prelimi-

previously

22).

The abbreviations used are: PA, pyrazoloacridine: topo. topoisomerase: TPT. topotecan: HPLC, high performance liquid chromatography; YPD medium. medium containing yeast extract. peptone. and dextrose (23).

-I

To whom requests Oncology Research,

displaced

agent.

PA

to

previously

of

studies

such been

( 1 1). lead

Leukemia

78229. 3

shown

examined

a mechanism

have

cells

II (e.g..

of these

of

induce

that

target

or topo

II inhibitors work

also

viscosity

agents have

breast

approaching

that

15)

can

( 12). PA also

a potency

enhanced

14 and

of

induction

events

PA was

intercalating

tumors

hepatoma

initial

concentrations

topo

PA

human

synthesis with

solid

in MCF-7

that

the

a result,

PA has been

with

and

and in situ

(20),

3B

As

the mechanism

of interest.

indicated

RNA DNA

different

of (6).

against

(8).

activity.

are unclear.

inhibit

bromide

of the

cells,

recent

cytotoxicity

to preferentially

short-

have

are

recently

activity

consistent

these

Despite

studies

cells

that

completed

fragmentation

an observation

activity

overexpression protein MRP

cancer

a matter

unique

4),

cells

displayed

Phase

of PA has become

cancer

basis of resistance

ovarian

of this potential

cytotoxicity

against

of PA were

II trials

antitumor

in noncycling

activity

PA

to

Preclinical

( 1,

cytotoxicity

trials,

acri-

several

selectivity

full

trials

these

(1-3).

displays

paclitaxel-resistant

is undergoing

II-

Cancer

Development.

( 1 , 4),

in P53-deficient

and

support was provided by NIH Grant and a fellowship from the Medical

of Canada (to M. C.). S. H. K. of America. address: Department ofClinical

Center.

(ROl

I

compounds

broad-spectrum

tumor

I clinical

one

intoplicine NIH

solid

agents on the (5) or the multidrug

and

clinically

1734

topo

but also I and topo

synthesized of

agent

PA

apoptosis

in drug-treated

Section

both

use.

class

has

I-DNA

12/5/97.

with

addition,

Recent

of this article were defrayed in part by the This article must therefore be hereby marked

adt’ertiseme,it in accordance indicate this fact. I Supported in part by grants

CA69912). Postdoctoral (to A. A. and M. C.)

accepted

agent

In

PA

Phase In

new

other

actinomycin

The costs of publication payment of page charges.

this

(4).

apoptosis.

that

Collectively,

target

a rationally

a

as an anticancer

inhib-

Consistent

indicated

8).

cisplatinPA

cytotoxicity of topotecan PA exposure was itself studies revealed that PA

as 250-fold

(7,

of

that

cells

resistant to P-glycoprotein

(9).

of topo

cells.

Moreover,

Clono-

stabilization

and in intact

in vitro

hypoxic

( I , 2).

strains

of covalent

etoposide-induced

II.

PA can

IA),

first

including

against

cells.

PA

vito

properties,

in yeast

Instead,

i,i

Two

nu-

was unaffected by selective loss of topo I or topo II activity. On the other hand, enzyme assays revealed that 2-4 pl PA abolished the catalytic activity of both topo I and topo II in vitro. In contrast to topotecan and etoposide, PA did not stabilize

topo

for clinical

Fig.

the

testing

indicate

yeast cell

of mammalian

PA

an

activity

I or II, mammalian

mammalian that

with

in noncycling and activity in Phase I

study,

samples

revealed

congener

is

clinical

studies

unknown

exceeded

that

I and that

licensed

366140;

derivative,

activity

acridine

suggest

presently

PA4 dine

ABSTRACT an

topo

INTRODUCTION

undergo

against hypoxic

not only

II poisons

Divisions of Medical Oncology [A. A. A.l and Oncology Research [P. A. S.. J. M.. J. M. R.. M. M. A.. S. H. K.l, Mayo Clinic: and Department of Pharmacology. Mayo Medical School [J. M. R., M. M. A.. S. H. K.]; Rochester, Minnesota 55905: The Johns Hopkins Oncology Center, Baltimore, Maryland 21287 [M. C.. E. K. RI; and Warner-Lambert/Parke Davis, Ann Arbor, Michigan 48105 IJ. S-L.l

(PA),

concentrations

to inhibit

and topo II at clinically achievable concentrations indicate that its mechanism is distinct from topo

H. Kaufmann3

Pyrazoloacridine

far

683

on DNA

resulting

A.

Research

111

Reid,

Sebolt-Leopold,

Scott

366140)

Charron,

Rowinsky,2

Jennifer

(NSC

Cancer

at Division of 2(X) First Street


684

Pyrazoloacridine

Effect

on

DNA

Topoisomerases

I and

A

II

B 0

0

JN394

.

JN394topl

0

CCH,5N(Cfl,5

I-

,,-

dH

JN394

I

C

,,

0

PA

H

C.)

Cl) 8) C

0 0

C.)

m-AMSA

Etoposide mM

0.75

0.5 mM

1.0 mM

0.5

PA mM

CPT 0.125 mM

m-AMSA 0.125 mM

Fig. I A, Structures of PA and amsacrine. B. effect of mutated topo II on PA sensitivity in S. cerevisiae. Parental and temperature-sensitive mutant strains were incubated at 26#{176}C or 37#{176}C for 15 mm, maintained at 26#{176}C or 37#{176}C during a subsequent 3-h treatment with drug or diluent. washed, and plated on YPD agar plates. After a 3-day incubation at 26#{176}C,colonies were counted. Data were expressed as a percentage of colony counts in diluent-treated samples. and U, parental JN394 cells and JN394top2-l cells containing atemperature-sensitive topo II allele, respectively. incubated at 26#{176}C. , the respective strains treated with drug or diluent at 37#{176}C. C, effect of topo I gene deletion on PA sensitivity. Yeast cells were incubated at 26#{176}C for 4 h with drug or diluent. washed, and plated on YPD agar plates. Colony formation was assessed 3 days later. , parental strain JN394: .. topo I-deficient strain JN394top I . Results are representative of three experiments.

MATERIALS

AND

Materials.

( 1 ).

Additional

tamed

the

was

a gift

etoposide

from

and

PA

as

doxorubicin

described

as

from

the

Sigma

topo

Division

(King

Chemical

I and

Co.

topo

All HPLC solvents were of PA in Yeast Strains.

from

Louis,

EDTA.

grade. isogenic

yeast

phis.

TN).

Yeast

were

(St. Jude

grown

kindly

and with

Hospital,

Mem-

gels

overnight

at 26#{176}Cin YPD

YPD

medium

At the with

and incubated

indicated water.

times,

serially

26#{176}C. Colonies contained

150-300

Cytotoxicity Logarithmically ing

5%

cillin A)

>50

microscope

ciency

on

of PA proliferating

fetal

with

diluent

-60%

Alternatively.

.0 at

10’ cells/mI

in

or diluent.

washed three times agar. and incubated at

Control

plates

typically

Myeloid Leukemia cells in RPM! 1640

bovine

streptomycin, at 37#{176}C with

cells

10-14

of

YPD

later.

in K562 K562

were days

counted

(dH,O) in these

cells

100 units/mb

serum,

at 200

later.

Cells. containpeni-

and 2 mri glutamine (medium the indicated concentration of

with medium A. Aliquots plated in 0.3% (w/v) agar

containing

treated

removed,

3 days

PA for 1 h or 24 h, sedimented once were

X

colonies.

100 p.g/ml incubated

were

were

plated

counted

heat-inactivated

0.

of 1-2

X g for 5 mm,

containing as described on an were

alone,

which

washed

1000-5000 cells (25). Colonies

inverted

Results

and

phase

compared

had

contrast with

Gels

ethidium

36

were

and

The

10%

by with

were Tris,

2 units

of PA (added

BSA.

of

extraction

mM

incubated

containing

from

reaction buffer consisting (pH 7.5 at 21#{176}C),0.5

photographed

of mi

reaction

(w/v)

SDS

was

and

an additional

2 ib

15-mm

1 : 1 phenob:chboroform

applied

30

mrvi

run at 45 V for 4.5

bromide,

of PA on topo

1 iil

To

(modified

was

067

mixture

SO pg/ml

samples

in

activity

plasmid

K, followed

chloroform,

II Activities.

concentration

of

proteinase

prepared

EDTA.

medium

.0, 0.5-1

at 26#{176}C or 37#{176}C with drug

aliquots

diluted,

were

0.5-I

=

and

addition

at 37#{176}C.After

II (24) were

24).

the

10 mg/mi

incubation

final

DY!’,

mM

Topo I catalytic

reaction

aqueous stock), and 50 mM Tris-HC1

by

oftopo

Late-log phase cells (absorbance 600 nm) were adjusted to a concentration (23,

0.5

Children’s

allele

Nitiss

I and

ng of supercoiled

the indicated

terminated

containing

a temperature-sensitive

400

Topo

of PA on topo

at 37#{176}C in a 20-p.l

a concentrated 100 mri NaCl,

of

by Dr. John

26),

suppliers:

strains JN394 (genotype inatA !SE2 rad52::Leu2 Topi Top2), JN394topl containing a topo I disruption (23), and JN394top2-l provided

ref.

of enzyme,5

Topogen

on

the effect

for 30 mm

(St.

of PA

investigate

Prussia,

II from HPLC The

of MD).

of

following

Effect

ob-

(Bethesda,

Beecham

from

were

Branch,

Institute

TPT

previously

amsacrine

Resources

human

(Columbus, OH). Cytotoxicity

well

Cancer

purchased

purified

as

SmithKline

were

and

MO);

of

National

Reagents

PA).

synthesized

Pharmaceutical

Treatment,

TPT

was

aliquots

from

Cancer

centration of PA or diluent for 1 5 mm, treated with etoposide or for 1 h as indicated. washed, and plated as described above.

METHODS

PA

to 1 % (w/v) NaH2PO4,

h, stained under

agarose 0.5

I msi i.g/ml

The

effect

and

with

UV

light.

decatenation was investigated using similar techniques, except that 400 ng of catenated trypanosomal kinetoplast DNA was used as a substrate, and reaction buffer was supplemented with 7.5 mi MgC1, and 1 mM ATP. Identical

Il-catalyzed

results

ments

were

scribed

in

were

obtained

performed Ref.

topoisomerase Effect

26)

or

activity. of PA on

nuclear

purified

topo

To

I-mediated

cleavable

supercoiled

plasmid

units

of enzyme

in reaction

with

and

reaction with

examine

I and

phenol:chloroform

was

topo

II as

of PA

formation incubated

for

buffer.

proteinase

when

experi-

(prepared

as

of

or TPT

in

vitro,

30

mm

After

chloroform

50%

of the

as

topo ng

effi-

were described

of the extracted above,

the indicated

con-

experiments.

were

incubated

with

5

One unit of topo

I relaxes


input

plasmid

of

at 37#{176}Cwith

termination

K, samples

and

on 400

cells

a cloning

de-

sources

Topoisomerase-DNA

effect

complex 067

SDS

the

assays

extracts

Drug-stabilized

Complexes.

50

in these

using

in this

assay.

Clinical

then 0.5

subjected

to electrophoresis

pg/ml

ethidium

effect of PA plex formation

or etoposide in

supercoiled

was

vitro

plasmid

purified

067

proteinase

30

mm

Band

Depletion

band

assay

depletion

micabby

growing

washed

once (pH

X

106

to 1 .5-mI

37#{176}Cwith

with

cells

microfuge

or

diluent

etoposide

was

tubes

and then

an additional

45 mm

(27).

monocbonal Dr.

Cheng,

15

(kindly

provided

by

and

in medium

K562

resuspended

A.

Cells

ice-cold

PBS,

Supernatants tions

were

of PA

curves

stored

in these

(0-10

Reverse-phase

10-p.m column The

flow

rate

3.2-mm

potassium 1 M KOH

at 450

on a Coulter

6

was

that

was

One unit of topo II decatenates

cells

(1500

guard

equipped beads CA).

the volume

with

effluent

stabilizing

topo

PA

kills of

of defined From the of the K562

200 ng of kinetoplast

DNA

dis-

distriimages

NIH

based

by of

Image

1.61

on the assump-

conditions

tivity

The

under

was

was

JN394topl

cells

cells

that

1 C).7

with

classical These

observations

drugs

under

essentially

et a!.

the

(24),

JN394top2-b

strain

In contrast,

bB).

PA

topo the

cells

II ac-

mechanism

solely

by stabilizing

topo

the effect

of this

in topo

I content.

parental

was

at

beast 1C). PA

in its mechanism

prompted

in greater

that

us

which

wild-type

(Fig.

1B, indicates poisons

reported camptoth-

cells, with

cells

As

I poison

in JN394topl

in Fig.

the

survival

by comparing

PA

the result

by

compared

Nitiss

distinguishes

contrast, in

cells whether

of diminished

compared

topoisomerase

of PA on topoisomerases

by

I gene,

In as

II have

kills

with

in the

a

in yeast

(26#{176}C) or

of the topo

diminished topo

cells

differ

the cytotoxicity

markedly

(Fig.

studied

amsacrine,

of amsacrine.

PA kills

was

(23),

a disrupted

diameter concentra-

that

that

some

of topo

incubation

clearly

that

clonogenic

at 37#{176}C(Fig.

from

these

Studies

we

present

of

target

determine

on

conditions

result

possibility

tam

toxicity.

This

of PA

To

amsacrine

was

and

amsacrine

(24).

used

functions

( 1 5- 1 8, 28, 29)

alleles that

previously

incubation

in yeast

1A).

or etoposide

by

1B).

PA

(Fig.

following II

reported

complexes

agent

the

strain topo

cytotoxic (Fig.

between

mechanism,

and

the

function

poison

identical

of amsacrine

abrogated

alter observations

complexes

an

(37#{176}C). As

ecin

analyzer

cells,

by

In the were

that PA might

evidence

etoposide,

where

remained

might

previous

II

Il-DNA

PA,

cerevisiae. techniques

temperature-sensitive

of the JN394top2-l absent

by

genetic

cells

effects

PA

similarity

contain strong

pH at a

determined

a channel

no

X intensity)

using

topoisomerase

topo

that

previously

particles).

fl) was

yielded subcellular

(area

cells

a

under

spherical.

that

alter

structural

provided

coupled of K562

cells

of complementary

suggested

agents

by the

I-DNA

acetonitrile:tetrahydro-

of the column

acquired

the and

two-dimensional

The possibility

well-characterized

M.

nm.

of PA in the cell extracts,

at 37#{176}C.

(E. diameter,

7-tim

were

the

calculations

the possibility

of cytotoxicity

and unknown RP-8

and

was

quantities of K562 cells.

inside

the

excitation

intensities

were

a number

cytotoxicity

standard

(adjusted to an apparent the solvents) delivered

Absorbance

of

were

(Chromtech)

diameter,

phosphate after mixing

volume counter

RP2

inside

times 1 ml

Concentrafrom

X 4-mm

of 20:5:80

and calibrated using polystyrene (Becton Dickinson, Mountainview, tion

analyzed.

a Lichrosorb

a Brownlee

consisted

mM

mean

in

of standard

on (250-mm

with

of 1 .0 mb/mm.

monitored The

X

phase

furan: 100 of 4.0 with

three

macromolecules.

calculated

separations

column

fitted

(15-mm mobile

were

accomplished

analytical

particles)

20#{176}C until

-

l06/mb

X

on ice, samples

to remove

X g

PA for 60 mm

washed

of

whole

cells

topoisomerases.

strains Dis-

at 200

agitation

incubation 5 mm

at

HPLC

were

Sciences)

1 mm,

vigorous

supematants

intercalating

Kiel).

of 2-3

the

and

using

for

of PA in Saccharomyces

study,

enzymes

Subcellular

p.M

from signal

performing and

Cytotoxicity present

mouse by

0-10

of

PA,

in PBS,

RESULTS

DNA

1hz

sedimented

nuclei

under as de-

or topo

PA) prepared by adding known extracts prepared from untreated

.LM

PA to methanolic supernatants

a 5-mm X g for

using

of

corresponding

and then

to examine

provided

and

the

software

1 mm,

School)

X g for by

X g for

I (kindly

with

bysed

After

at 12000

for

topo

were

treated

and

-20#{176}Cmethanol. sedimented

incubated

Medical

cells

at 3200

at 37#{176}C,sedimented

were

at a concentration

were

of

or

University

Images

estimates

derived

integrated

tion

that

laser

cells

10 J.M

of

hydrochloride electrophoresis

Kelbner,

were

K562

microscope

argon-krypton

the

of PA.

resuspended

confocal

autofluorescence

cellular

at 37#{176}Cwith

PBS,

filter.

mean

685

calculated.

60 mm

310

emission

the

Research

added

37#{176}C.TPT

(27)

and

LSM

Quantitative

of PA

comparing

for ice-cold

an

where

nuclei

10 mM were

at 3200

of PA Accumulation

Log-phase

for 5 mm

with

Udo

logarith-

with

from

signal.

bution

a

at

performed

University Dr.

Measurement tribution.

was

topoi-

times

sample,

was

distribution

incubated

on a Zeiss

cernibbe

concentrations

Cells

three

conditions

sedimentation,

abiquots

mm

as indicated.

that recognize

Yale

bong-pass

at a concentration

at 37#{176}C, sedimented

antibodies

Y-C.

by

varying

for

Immunoblotting

590-nm

containing

One-mb

immediately solubilized in 6 M guanidine reducing conditions, and prepared for scribed

band

In brief,

containing

added

488-nm

27).

B.

incubated

and

in Ref.

7.4 at 20#{176}C)] and resuspended in medium

examined

using

in the

the subcellular

A were

mrvi

of covalent

1640

To examine in medium washed

present

of PA in the cells

with 7.5 with SDS

examined

harvested

of cells

of

was

were

number

units

cells

B [RPMI

comng of

in the presence above.

The formation

medium

4

the

concentration

the

phenol:chboroform

ebectrophoresis as described

(reviewed

cells/mb

of

approach,

supplemented the reaction

in intact

K562

with

at

samples

Assay.

adducts

HEPES

the

and performing ethidium bromide

somerase-DNA

in the presence

a similar

on topo Il-mediated cbeavable examined by incubating 400

for

K, extracting

and chloroform, of 0.5 p.g/ml

PA

Using

topo 116 in reaction buffer and 1 mM ATP, stopping

MgCl2

1-2

in the dark

bromide.

Cancer

to examine

conJN394

as

toxic

This

in

result,

differs

from

of cytothe

effect

detail.

and

in 30 mm

7 The enhanced cytotoxicity of amsacrine and etoposide after topo I disruption (Fig. 1C) has been noted previously has been attributed to increased reliance on topo II during these cells (44).


in yeast cells (42. 43) and replication in

686

Pyrazoloacridine

Effect

on DNA

Topoisomerases

A

I and

PA

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2 Effect of PA on topoisomerase catalytic activity and formation of topoisomerase-DNA covalent complexes. A, inhibition of topo I activity. Supercoiled plasmid was incubated with 2 units of topo I in the absence (Lane 2) or presence of PA (Lanes 3-8) or doxorubicin (Lanes 9-14) at a final concentration of0.25-8 p.M as indicated. Lane 1, substrate DNA incubated without enzyme. SC, supercoiled DNA; R, relaxed DNA. B, inhibition of topo II activity. Kinetoplast DNA was incubated with 2 units of topo II in the absence (Lane 2) or presence of PA (Lanes 3-8) or doxorubicin (Lanes 9-14) at a final concentration of 0.25-8 p.M as indicated. Lane 1, substrate DNA incubated without enzyme. N, kinetoplast network DNA. MC’, released minicircle DNA. Results are representative of four experiments. f, stabilization of topo I-DNA complexes by TPT but not PA. Aliquots of supercoiled plasmid 067 were incubated for 30 mm at 37#{176}C with nuclear extract containing 50 units of topo I in the absence or presence of TPT ( I .56 p.M) or PA (200 p.M) as indicated. After treatment with SDS and proteinase K, samples were separated on I .0% agarose gels in the presence of 0.5 p.g/ml ethidium bromide to better separate nicked (N) from relaxed (R) DNA. Note that TPT stabilizes topoisomerase I-DNA complexes (as indicated by the increased amount of nicked DNA), whereas PA does not. D, stabilization of topo Il-DNA complexes by etoposide but not PA. Aliquots of supercoiled plasmid O were incubated for 30 mm at 37#{176}C with purified topo II in the absence or presence of etoposide (400 p.M) or PA (500 pM) as indicated. then treated with SDS/proteinase K and subjected to electrophoresis as described for C’. Note that etoposide stabilizes topoisomerase-DNA complexes (as indicated by the increased amounts of nicked and linear DNA), whereas PA does not. E and F, PA inhibits Fig.

stabilization concentration

presence j.g/ml

of topoisomerase-DNA of PA along with

of 400 ethidium

p.M

etoposide

bromide.

PA Inhibits

Topoisomerases

In a complementary topoisomerase When 14,

series activity

topo

I catalytic

of

a supercoiled

version Lanes

The

1 and

concentrations

2,

as

14.

Lanes

under

was examined activity plasmid

as

2

JO and

Ii).

pM

cell-free

assayed

to relaxed PA (Fig.

DNA this

5).

topo

II catalytic

by monitoring

release

network

of kinetopiast

DNA

inhibition

was

observed

of free (Fig.

DNA

at 4 p.M PA

minicircles

Lanes

2B,

(Fig.

2B,

from

1 and

2),

Lane

4).

>80%

Doxo-

rubicin

(Fig. at

centrations of 2 p.M or more (Fig. 2B, Lane 1 1). These observations indicate that PA inhibits both topo I and topo II catalytic

to

activity. To examine

Doxorubicin,

activity

assayed

con-

activity

binds to DNA with affinity similar inhibited topo I activity at 2-4 When

on

conditions.

circular

Lane

of PA

by following

inhibited 2A.

Conditions.

the effect

under

was

respectively),

low

Cell-free

of experiments,

an intercalating agent that that of PA (13), likewise (Fig.

complexes. Aliquots of supercoiled plasmid 067 were incubated for 30 mm at 37#{176}C with the indicated nuclear extract containing 50 units of topo I in the presence of 6.25 p.M TPT (E) or 4 units of purified topo II in the (F). After treatment with SDS and protemnase K, samples were separated on 1.0% agarose gels in the presence of 0.5 portions of gels containing nicked (N) and linear (L) plasmid are shown.

p.M

was

similarly

all DNA-targeting endonucleases

inhibited

topo

the possibility enzymes, (EcoRI,

NotI,

Il-catalyzed

that

decatenation

PA nonspecifically

at con-

inhibits

the effect

of PA on three

restriction

and

and viral

ligase


SfiI)

DNA

was

a

Clinical

A

Cancer

687

Research

3000

ic

0 C 0 C)

10

In

a) C 0 0

0 4

8

(h)

Time

50

75

(uM)

Concentration

100

2

0

4

6

8

0

10

2

PA (tiM)

6

4

Extracellular

8

10

PA (tiM)

j’ =

;LL; Fig.

3

Assessment

or doxorubicin,

of PA cytotoxicity

then washed

and

and plated

accumulation

in drug-free

in K562

concentration of PA, then washed and plated in drug-free indicated concentration of PA, then washed and extracted and Methods.” Inset, retention of PA in K562 cells after by confocal microscopy. Results are representative of

investigated. decrease able,

In assays

performed

in restriction concentrations

and

no effect

up to 10

had

or Nod.

no effect

short,

PA

inhibited

topo

those

that

affected

other

PA Does

Not

Additional

mine

whether

the

results

from

topo that

the

complexes,

separate

subjected

nicked

I poison

TN’

detected

in this assay

ing

topo

interruption

I-DNA

of the

complexes

(Fig.

2C,

are

formed.

topo

Il-DNA

incubated

Topoisomerase-DNA

subjected

to ebectrophoresis,

below

performed

to deter-

catalytic

to stabilize was

incubated

with under

(Fig.

backbone

In contrast,

Lane

with

and

drug,

of nicked

failed 2).

10 PA

with

purified with

2C,

the results

between

topo SDS

Lane

3),8

concentrations

(Fig.

topo

and

were

again

0.5 and 500

proteinase Lane

and

K. 3),

and the

reflect-

complexes

respectively. linear

to

increased

2D,

Il-DNA

nicked

of PA supercoiled

II in the absence

and

II dimer,

ability

When

markedly

plasmid

of the topo

the

2D).

etoposide

and linear

to increase

to examine (Fig.

of covalent

sites

Identical

concentrations

used

treated

stabilization

active

(Fig.

using

at one

In contrast,

species

obtained

p.M. Collectively.

(Fig.

using the

2D,

10 PA results

conditions

2C).

The topo

of nicked

Lanes

PA

I-DNA SDS

the

or both

comtopo

amounts ing

activity

treated

plasmid

phosphodiester

was

complexes

stabilize

plasmid

p.M.

strategy

of

the amount compare

500

nicked

obtained

was

to electrophoresis

increased

of

were

and

0.5

A similar

of PA In

amount

results

presence

of PA,

and supercoiled

markedly

the

between

treated

In B, K562

plasmid

plasmid

and presence

increase

on Sf1

not shown).

were

enzymes. were

of PA

cells

survival.

Identical

topoisomerase-DNA

supercoibed

K, and

(data

of topoisomerase ability

effect

a 50%

II at concentrations

topo

of covalent

I in the absence

proteinase

ligase

experiments

stabilization

little

In A, K562

clonogenic

detect-

concentrations

Covalent

inhibition

To examine

covalent

I and

where

be readily

had

p.M

DNA-targeting

Stabilize

Complexes.

plexes.

would

Likewise,

on DNA

for 1 h with the indicated concentration of PA cells were treated for 24 h with the indicated agar to determine clonogenic survival. Bars, SD. In c, K562 cells were incubated with the so that average cellular PA concentrations could be determined as described in “Materials removal of PA from the extracellular medium. D, subcellular distribution of PA as assessed three experiments.

conditions

activity

of PA up to 50

on EcoRI

p.M

under

enzyme

cells.

agar to determine

4 and when

PA

plasmid 5),

S

reflectcovalent

failed

The

altered

migration of the supercoiled DNA in Lane 3 reflects the relaxation of DNA in the presence of the intercalating Similar results have been described previously with other agents (38).

topo I-mediated to

agent PA. intercalating


688

Pyrazoloacridine

Effect

on DNA

Topoisomerases

I and

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Fig. 4 Effect of PA on cleavable complex stabilization and cytotoxicity of etoposide and TPT. A, effect of PA on topo I-DNA complexes in intact K562 cells. Cells were treated with 0.5% DMSO (Lanes 1-4), 3. 1-50 p.M TPT (La,ies 5-9), or 1-500 p.M PA (Lanes 10-15) for 45 mm, then lysed under denaturing conditions and prepared for SDS-PAGE. Gels were loaded with polypeptides from 0.3 X l0 cells (Lane I) 0.75 X l0 (Lane 2), I .5 x (Lane 3). or 3.0 x l0 cells (Lrnies 4-15). After electrophoresis. samples were transferred to nitrocellulose and probed with anti-topo I. B. band depletion assay for topo II. Cells were treated with 1% DMSO (Lanes 1-4), 34-680 iM etoposide (Lanes 5-9), or 1-500 M PA (Lanes I0-/5 for 45 mm, then lysed under denaturing conditions and prepared for SDS-PAGE. Gels were loaded with polypeptides from 0.3 X l0 cells (Lane 1). 0.75 X l0 cells (Lane 2). 1.5 x l0 cells (La,ie 3), or 3.0 X l0 cells (Lanes 4-15). After electrophoresis, samples were transferred to nitrocellulose and probed with an antiserum that recognizes topo lbs. Similar results (band disappearance with etoposide but not PA) were obtained when blots were probed with an antiserum that recognizes topo bIn and topo II. C’, effect of PA on TPT-induced stabilization of topo I-DNA covalent


Clinical

presented

in Fig.

2, c and

D, indicate

covalent topoisomerase-DNA Instead, PA actually topoisomerase-DNA when

complexes

nuclear

and

TPT

extracts

in

treatment

the

II was

in the presence resulted These

4-14).

licensed

clinical

quent

were cells:

in cytotoxicity,

range

that

inhibit

At

are

of topoisomerase-DNA

issues,

the

med

in K562

human

of its high

In contrast,

and

leukemia cloning

cells

agar,

cells.

when

were

incubated

cells

were

biochemical experiments. To determine the extracellular 1 h with

was

loss

of

PA was

cally ated

PA

from

fluorescent, drug

experiments

cells

stably

exceeded

bound

ques-

reflects

the

that

molecules

in the

treatment

affect

the

etoposide,

and its

detectable

then

results

3A).

stabilize

PA,

agent (8). These observations that were explored in further PA

levels

K562 PA,

that

cells

accompany

were

extensively

that

by

cells.

3C,

distribution was

cells.

inset), PA

just

vesicles confocal

it had

that

the

tions,

for

PA

Likewise,

I

decrease 4B,

Lanes

contrast

in 5-9),

as they

to TPT

of concentrations

failed

cell-free

TPT-induced was

and

to cause

4F)

the in

these

that

under

cell-free

I).

fashion

topo

of TPT

as bow as is readily

ad-

1.25 in Fig.

the action

4E) effect

in

and was

(Fig.

p.M

4F,

patients

4, C-F,

of topo

similar

present

observa-

(Fig.

surpassed

the results in a manner

the

Il-DNA these

inhibitory

This

Likewise, in the

with

cells.

of altering cells

assays

fashion

topo

cytotoxicity

PA (8). Collectively, mammalian

depletion

5-I

Consistent

concentration

PA is capable

topoisomerases

Lanes

to cells

of covalent

of covalent

to

cells.

etoposide

in a dose-dependent

4C,

5-11).

fails

in intact

Band

in a dose-dependent

diminished

and

stabilization

(Fig.

Lanes

at a PA

that

conditions.

stabilization

PA

in intact

complexes

diminished

of PA

4D,

that

of TPT

the ability

a concentration

inset),

suggest

inhibited

the

(Fig.

II in intact

These

of topo

to DNA.

1hz molecules In

observations

under

also

observed

date

DNA.

is

to 48#{176}C(30),

(Fig.

of topo to

4A,

which

numbers

complexes

diminished

(Fig.

etoposide

receiving

bound

topoisomerase-DNA

presence was

by PA

heating

topoisomerase-DNA

complexes

I-DNA

indicating

throughout

PA

as

of PA

(Fig.

100,000,

170,000

range

these

covalent

revealed

ducts

of the cell-associ-

distributed

13),

covalent

Instead,

is intrinsi-

microscopy.

at a wide

in a dose-depen-

in a dose-dependent

mobility bound

or

decreases in the signals for topo I or topo II (Fig. 4, B, Lanes 10-15). Coupled with previous alkaline elution

stabilize

and

After

the half-time

Because

by confocab PA

K562

the medium,

altered

results

TPT

with

l00000

of increasing

1hz at Mr

etoposide-induced

incubated washed,

the

(10,

the

to the 1 p.M for >24 h

etoposide topo

colony-forming

results

brief

covabently

PA cells,

PA.

Mr

by

whether

cells

at Mr at

is reversed

mobility

for

PA

A and

(Fig.

24 h with

with

TN’

signal

become

covabently

1 h and

for

altered

with

the

II in intact

and of

of

I signal

of

I and

as they

reflecting

chosen

assays.

topo

signal

was

loss

(27)

presence

cells topo

within

conditions.

Topoisomerase-

topo

treatment

and

of intact in the This

for

observed

cell but was concentrated in perinuclear (Fig. 3D). Estimates derived from these gested nuclei,

5-9).

PA for

8 h (Fig.

within

be determined

indicated

Lanes

with

250-fold

PA from

the subceblular

could

decrease

I and

assays

after

well

To determine

topo

depletion

absence

Treatment dent

are

of PA

cell-free

of Covalent

with

performed in the

line

(-60%)

band

were

become

agar

of interacting

etoposide

by

approximately

capable

that

I and topo II under Cells.

in

concentrations

concentrations

in Intact

intranuclear

the experiments

extracellubar

the Stabilization

Complexes

p.M was

incubated

intracellular

of extraceblubar

that

PA Alters

exam-

This latter value is close that is sustained in patients

p.M extracellular

concentrated

topo

to inhibit

specific

extracted so that cellular PA content could be determined HPLC. Results of these experiments (Fig. 3C) indicated that removal

PA

were

depletion

cytotoxic

in intranuclear

of PA

cell

that

689

Research

PA concentration

in an average

Collectively,

p.M.

shown

DNA

to result

of -200

range

PA

To address

an extracellular

result

Subse-

PA

3B).

concentrations,

1-10

Does in situ?

in soft

3 indicate

assays

of

of PA

(b)

This

Fig.

topoisomerase

presently

concentrations

band

of -50

after i.v. administration of this provide the range of concentrations

for

effects

Cells.

cytotoxicity

concentration

PA Lanes

the following

vitro?

efficiency

an IC50

IC50 was 1.25 p.M (Fig. PA serum concentration

these

the

2F,

poisons

PA

for subsequent

K562

in soft

that

complexes

accumulation

of manipulation When

(Fig.

concentrations

in

PA

was

in etoposide-

Leukemia

intracellular

observations, be expected

and

concentrations,

to address

topoisomerases

stabilization

plated

when

plasmid

species

extracellular

on these

of 1 p.M would

in the amount

decrease

Human

designed

(a)

these

ease

PA

use.

result

because

PA

indicate

plasmid

Likewise,

of the topoisomerase

of PA on K562

in intact

example,

concentrations,

supercoibed

linear

again

those

experiments

tions

and

of covalent For

4-14).

of increasing

observations

from

Effect

Lanes

Based

stabilize

supercoibed

PA

with

of nicked

are different for

with

in a dose-dependent

formation

not

decrease

2E,

incubated

treatment

does

agents.

increasing

(Figs.

etoposide induced

incubated of

nicks

topo

PA

by other

in a dose-dependent

of TPT-stabilized purified

were

presence

resulted

that

complexes. inhibited the stabilization

Cancer

mdi-

I and

topo

to its effect

on

conditions.

the

and in nuclei images sug-

that 23 ± 5% of the total cellular PA was detectable in which occupied 27 ± 5% of the intracellular volume.

DISCUSSION Despite synthetic

the promising

acridine

derivative

preclinical PA (see

activity

displayed

“Introduction”),

by the the mech-

complexes. K562 cells were treated with 12 p.M TPT in the presence of 0-400 p.M PA (Lanes 5-1 1) for 45 mm, then bysed under denaturing conditions and prepared for SDS-PAGE followed by immunoblotting with anti-topo I. Lanes 4-11 were loaded with polypeptides from 3.0 X l0 cells. Lanes 1-3 contained a serial dilution of diluent-treated cells as in A. D, effect of PA on etoposide-induced stabilization of topo Il-DNA covalent complexes. K562 cells were treated with 68 p.M etoposide in the presence of 0-400 p.M PA (Lanes 5-li) for 45 mm, then lysed under denaturing conditions and analyzed as in C. The blot was probed with anti-topo 1hz. E and F, clonogenic assay comparing effect of a 1-h treatment with TPT (E) or etoposide (F) in K562 cells that had been preincubated for 15 mm in the absence (-) or presence (---) of 25 p.M PA. Note that PA abolishes the cytotoxicity of etoposide and TPT without having any toxicity under these conditions. inset, cbonogenic assay comparing survival when cells were preincubated for 15 mm in the presence of the indicated concentration of PA before 17 p.M etoposide was added for an additional 60 mm. Bars, SD.


690

Pyrazoloacridine

anism

the

of

Effect

cytotoxicity

present

work,

topo

I and

both

stabilization

of

we

of PA

on

effects

of the

topo

in vitro

and

of a potential effect

altered has

on

studies

plexes

and

reports

have

plexes,

including

1) rube out

mechanism could

might

Il-DNA

identified

agents

quinolinediones

(21),

stabilize

( 19),

and

however,

rule

both

intoplicine

possibly

types

(20),

actinomycin

of

the

tion

16).

activity

PA

not involve

other agents that inhibit topoisomerase stabilizing covalent enzyme-DNA scribed, and

including

ICRF

chone from

the

193 (32)

(Ref.

33;

these

for

in

agents I-directed

view,

The

complexes appears cited agents. observations

I and topo intact

cells.

First,

ability

to distinguish

PA

relatively

rapid

trations

enzymes

PA

toxicity

is also

involve plexes. (Fig. when

stabilization In contrast and incubated

toxicity bution

(Fig.

in nuclei

D).

2, A and

Based

survive activity,

some

period

eukaryotes

such

The contrasted not

inhibit

topo

potencies

as

results

those

only

bind

I and

(Fig.

yeast

not

cometoposide

K562 of

might

be

the

inhibition

the

PA the

(8). also

present

of topo

of cleavable

cytotoxic

within

PA might

metabolism,

that

to the

PA concen-

receiving that

(Fig. these

damage.9 are

in patients

stabilization and

vitro

lethal

experiments

possibility

pertinent in

of PA through

extracellular

in DNA

upon

of doxorubicin observed after

acting

out the possibility

without

highly

PA exposure

present

involved

raise

II activity

Drs.

Theresa

Shapiro,

of John

Hilton

Deb Strauss,

I and

complexes

actions

of

prolonged

in viva.

Randall

and and

John

James

K. Johnson, Nitiss

Tarara,

are gratefully

Udo

for kind

gifts

as well

Kellner,

Y-C.

of reagents.

as the secretarial

The

Cheng, advice

assistance

I. Sebolt,

J. S.. Scavone,

2. Jackson, R. C., pyrazoloacridines: selective cytotoxic

S. V., Pinter,

Rev.

C. D.. Hamelehle.

Pyrazoloacridines, against solid tumors

K. L.. Von

a new in vitro.

class of Cancer

Sebolt, J. S., Shillis, J. L., and Leopold, W. R. The approaches to the development of a carcinomaagent. Cancer Invest., 8: 39-47. 1990.

S. L. Status Crit.

C.

of new anthrapyrazole Oncol.

Hematol.,

22:

and pyrazoloacridine 79-87,

topoisomerase

J., Havlick, M.. Hamelehle, K., Nelson, J., Leopold. W., R. Activity of the pyrazoloacridines against multidrug-resistant cells. Cancer Chemother. Pharmacol.. 24: 219-224, 1989.

6. Cole, small

cell

250-256.

enzymes

de-

1996.

4. LoRusso. P., Wozniak, A. J.. Polin, L., Capps. D.. Leopold. W. R., Werbel, L. M., Biernat, L.. Dan, M. E., and Corbett, T. H. Antitumor efficacy of PDI 15932 (NSC 366140) against solid tumors of mice. Cancer Res., 50: 4900-4905, 1990.

tumor to

of

acknowledged.

REFERENCES

Jackson,

4, C and

are able

these

topo

rule

enzymes

is not

p.M

observed

we cannot other

is observed

of inducing 1-10

of the

presumably

concentrations PA

forma-

5. Sebolt,

II in vitro

cells

just

3B),

accudistri-

(Fig.

diminished

(Fig.

PA

topo

of the

that

concentrations of cytotoxicity

that

the

(16),

at the level

mechanisms

killing

mechanisms

of concentrations

3. Berg. rivatives.

concentrated

I and

inhibition

S. P. C. Patterns

of cross-resistance

lung carcinoma 1990.

line.

Cancer

and

in a multidrug-resistant Chemother.

Pharmacol..

26:

is

as it is in lower

(35).

with

to DNA

topo

does

subcellubar

in situ

that

with

obtained

PA with

with

II catalytic

2, A and

that

is rapidly

enzymes

toxicity

obtained

with

agents

of time

with

affecting

by poor

topo

it appears

prolonged

associated

of

view

that

effects

other

cell

Hoff, D. D., and Jackson, R. antitumor agents with selectivity Res., 47: 4299-43()4, 1987.

for PA-induced

and PA

inhibit

these

results,

although

obviously

that

readily

B) and affect

on these for

explained

accumulation

3, C and D) indicate that

and

in intact

topoisomerase-DNA as TPT (Fig. 4E),

is not

of drug

to levels

TPT-

is capable

course

of of PA

(Fig. 3A), PA is relatively nontoxic for 1 h (Fig. 3A). This relative back of

exposure

Studies

PA

a mechanism

of covalent to drugs such

of a I-h

mulation.

decrease

intercalating

of doxorubicin

complexes

possibly

micromolar

the

in most

We thank of

inhibition

complexes

the time with

doxorubicin with cells

4F),

(Fig.

consistent

to

of

ACKNOWLEDGMENTS

all of the

to the cytotoxicity

of

Second,

in situ.

classes

from

used

observations

differs

topoisomer-

that the catalytic

ability

3-lapa-

PA

both

covalent

etoposide-stabilized topoisomerase-DNA cells (Fig. 4, C and D) indicates that these

34),

I and

relatively

(31)

agent

to inhibit

also contribute the

Ref.

stabilizing

suggest

II might

see

without been de-

merbarone

extensive

with

Although

catalytic

activity have

Il-targeted

without

in vitro

Several

the

catalytic intermediates

as the topo

alternative

as well.

topoisomerases

topo

topo

as well

agents

ase-DNA previously

inhibits

I (Fig. 14) and topo II (Fig. 2B) by a process that stabilization of cleavage complexes. Although

oftopo

does

Instead,

to the

to low

inhibit

13).

effects

cytotoxicity

Il-DNA

and

(4 1 ). These

is consistent

(Fig.

10 and

membrane

3A)

range

Refs.

(40),

a I -h exposure

contrast to these agents, PA does not appear to stabilize topoisomerase-DNA complexes in vitro (Fig. 2) or in intact cells 4;

to the the

of topo

radicals

It is noteworthy

In

The

II enzymes on DNA (20 and 36 and 37) might make these

exposure to submicromobar 3A). In contrast, the paucity

indobo-

D (14,

stabilization

of free

other,

com-

sensitive Nonetheless,

topo

(15).

differs from that of PA (Fig. 3A). Doxorubicin has been to have other effects that contribute to its cytotoxicity,

the

Previous

inhibiting

clearly shown including

39).

be

into DNA

of topo I and topo respectively; Refs.

particularly

brief (Fig.

com-

might

(38,

contribute

out

I-DNA

doxorubicin,

of intercalation

agents

PA

campto-

like

II as a result

plasma

that or

topo

in

containing

simultaneously.

that

saintopin

to be

presently

strains

covalent

complexes

enzymes

II appears

amsacrine

not,

stabilize

the

compound.

the possibility

as

large footprints 28 nucleotides,

of

in yeast

PA,

the

(c)

etoposide the broad

drugs

that

without

II poison provide

topo

bility

topo

and

for this

I and

In

inhibits

in abrogation

or the topo observations

topo

studies

PA

topo

occurs

mechanism

(Fig.

yeast that

PA

(a)

inhibition

performed

cytotoxic

uncertain.

that:

of topoisomerase-directed

The

These

possibility

remained

results

These

PA

II

complexes;

cytotoxic

of

same

thecin.

has

this

cells.

topoisomerases the

(b)

TPT

that

use.

II;

I poison

in intact

from

agent

I and

demonstrated

topoisomerases

outline The

this

topoisomerase-DNA

effect

different

Topoisomerases

have

topo

of

clinical

on DNA

B). These

are

to

be

compared

doxorubicin.

similar

affinity

activity

observations

and

These (13)

in vitro

raise

with

two

but

also

similar

the possi-

indicated in Fig. 3A, extremely high concentrations are associated with cytotoxicity. even with exposures 1 h. The present studies have not addressed the mechanism

9

As

p.M)

acute toxicity different

target

and cannot at these

rule out the possibility very

high

concentrations.


of PA (>25 as short as of this more

that PA acts through

a

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