The formation and resealing of intercalator-induced DNA strand breaks in isolated L1210 cell nuclei

Vol. 107, No. 2, 1982 July 30, 1982

BIOCHEMICAL

AND RESEALING BREAKS

Pommier,

RESEARCH COMMUNICATIONS Pages

THE FORMATION

Yves

AND BIOPHYSICAL

Donna

IN

OF

INTERCALATOR-INDUCED

ISOLATED

Kerrigan,

L1210

Ronald

CELL

DNA

576-583

STRANIO

NUCLEI

Schwartz

and

Leonard

A.

ZwellingI

Laboratory of Molecular Pharmacology Developmental Therapeutics Program Division of Cancer Treatment National Cancer Institute National Institutes of Health

Received

June

4, 1982

DNA single-strand breaks and DNA-protein crosslinks induced by intercalating agents were measured in mouse leukemia L1210 cell nuclei by the alkaline m-AMSA and 5-iminodaunorubicin produced protein-associated elution technique. DNA breaks but at a lower level than that produced in whole cells. The frequency of drug-induced DNA single-strand breaks and DNA-protein crosslinks were approximately equal. The rapid formation and resealing of DNA breaks produced by m-AMSA were similar to that seen in whole cells. The DNA-protein crosslinks The process by which reversible, were also reversible after m-AMSA removal. protein-associated DNA strand breaks are produced by intercalating agents can now be studied in an isolated chemical system possessing the breakingrejoining capacity of whole cells. INTRODUCTION Various DNA

appear

to

be

following

form

located

qualities

of

effects

use of

close the

of

ITo whom correspondence 5D17, 9000 Rockville Abbreviations used: crosslinks; m-AMSA, 5-ID, 5-iminodaunorubicin;

agents

Pike,

DNA-protein

shown

may

to

produce

crosslinks

(DPC)

stoichiometrically

each

other

treatment,

in

and

the

cellular

DNA

response

has

represent

led

to

the

enzymes

such

reversible in

whole

cotemporally (3,

produced hypothesis

cells

6). in

that

as

topoisomerases,

to

study

The whole these

(2-6).

nuclei,

intercalating

been

breaking-rejoining

DNA scissions

isolated

have

reverse to

DNA

proteins the

and and

intercalator

generate The

(SSB)

2 effects

DNA-break-associated which

agents

breaks

These

enzymatic cells

intercalating

single-strand

(l-7). and

DNA

instead would

of

whole

minimize

should be addressed at Rethesda, MD 20205.

cells, drug Building

interactions 37,

with Room

SSB, single-strand breaks; DPC, DNA-protein 4'-(9-acridinylamino)-methanesulfon-m-anisidide; 2-Me-g-OH-E+, 2-methyl-9-hydroxyellipticinium.

0006-291X/82/140576-08$01.00/0 Cop,vrighl @ 1982 hv Acodemrc Press, Inc. A II righrs of reproducfion in any form reserved.

576

the

DNA cyto-

Vol. 107, No. 2, 1982 plasmic

constituents

nature

of

an

isolated

the

ability

nuclei

the

chemical to

bility

were In

the

different

system.

the

Filipski

and

Kohn

the

formation

not

studied. study

intercalating

these of

effects

we

describe in

be

a system

previously

produce

DNA

could

such

(personal

these

nuclear

agents

utility

of

drugs

to

RESEARCH COMMUNICATIONS

described

effects

the

and

DPC

and

more

DNA

in

isolated

isolated

L1210

of

derivative

derivative

5-iminodaunorubicin

the

(5-ID),

breaks

in E -ID

whole

for whole in

as

cells

(3-5).

isolated

cells.

nuclei.

study

By Of

were

in

isolated

nuclei

responses

elicited

MATEPIALS

AND METHODS

by

Cells and radioactive a suspension culture previously described

these

that

the

the

similar did

can

labeling: in RPM1 1630 (3).

not

both be

reversi-

mouse plus

the

3 agents

in

an

those

and isolated

leukemia 15% (v/v)

cells fetal

by

produced in

DPC were

breakage

DNA

produced

breaks

SSB and

the

studied

L1210 medium

to

the

ellipticine

DPC

produce

m-AMSA

suggesting

and

one

but

protein-associated SSB

qualitatively

however,

drugs

that

chemically

These

reversible,

2-Me-q-OH-E+

importance,

rever,sible

produce

We found

nuclei

greatest

all

the

9-anilinoacridine

(2-Me-q-OH-E+).

they

contrast,

and

in

found

(m-AMSA),

2-methyl-9-hydroxyellipticinium chosen

cells

their

three

nuclei;

upon

nuclei,

critically

effects

the in

depend

have

in

Thus,

would

communication)

SSB

(7,8). characterized

4'-(9-acridinylamino)-methanesulfon-m-anisidide

anthracycline

in as

The

can

access

by

all

present

derivative

drug

produced

ellipticine, of

AND BIOPHYSICAL

maximize

reproduce

system.

kinetics

and

and

DNA effects

intercalator,

were

BIOCHEMICAL

m-AMSA in

isolated rapidly

rejoining chemical

were calf

system.

grown serum

L1210 mouse leukemia cells were centrifuged Isolation of L1210 cell nuclei: and resuspended in an equal volume of nuclei buffer (150 mM NaCl, 1 mM KH2P04 5 mM MgC12, 1 mM EGTA, 0.1 mM dithiothreitol, pH 6.4) at 4°C. These cells were again centrifuged and resuspended in l/10 volume iced nuclei buffer. Then 9/10 volume of nuclei buffer containing 0.3% Triton X-100 was added and the mixture incubated for 10 min at 4'C. The nuclei were then pelleted by centrifugation (1200 rpm for 5 min) and resuspended in nuclei buffer at 37". Nuclei were observed microscopically as trypan blue positive. Additionally electron microscopy of these nuclei revealed the disruption of the cell membrane, the depletion of the cytoplasmic contents and the preservation of the nuclear membrane. Membrane tags, however were still present. (Electron microscopy was performed by Dr. Lance Liotta, Laboratory of Pathology, NCI).

Drug

Drugs and Synthesis

drug and

treatment: Chemistry

m-AMSA base Branch, Division

(NSC of

249992), Cancer

obtained Treatment,

from the National

Vol. 107, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Cancer Institute, was dissolved in 100% dimethyl sulfoxide at 10 mM and stored frozen. 5-iminodaunorubicin (NSC 254681) was a gift from Or. Robert I. Glazer, Applied Pharmacology Section, Laboratory of Medicinal Chemistry and Biology, National Cancer Institute who obtained the compound from Dr. E. Acton, Stanford Research Institute. 5-iminodaunorubicin was dissolved in Z-methyl-9-hydroxyellipticinium glass distilled water at 1 mM and stored frozen. acetate was a generous gift from Dr. J. B. LePecq, Laboratoire de Pharmacologic Moleculaire au CNRS, Institut Gustave-Roussy, Villejuif, France. Stock drug was dissolved in glass distilled water at 8.25 mM and stored frozen. All Treatments were stopped by a 1:30 drug treatments were for 30 min at 37'C. dilution of treated nuclei in drug-free nuclei buffer in the studies of disappearance of drug-induced DNA SSB and DPC. Alkaline elution: SSB and DPC were quantified in nuclei by alkaline as previously described for whole cells. Drugs were removed from just prior to elution by a 1~20 dilution in iced nuclei buffer. of methodology and quantification of SSB and DPC using 3H-DNA-labeled standard cells have been given in previous publications (3-7, 9).

elution nuclei Details internal

RESULTS DNA from

SSB

isolated

nuclei

eluted (Figure

than

1 break

accurate

nuclei

slightly

lo7

DNA

of

breaks

were

DNA

cells

but

dose

range

the

there used

isolated

alkaline

nuclei in

elution (9).

This the

no

(Figure

close

(Figure isolated assay

is

identical association

from

a 30 min

up

to

2

with

isolated

nuclei

apparent

m-AMSA

becoming

cells at

saturation

2

Y

in

isolated

limited M.

(Figure

in

The

that

frequency

2A).

a lower in

no more

agents.

above in

whole

allowed

intercalating

treatment

than

R irradiated

produce

and

concentrations less

to

M thereafter

r

300

treated

appeared

by

DNA

and

Rad-equivalents)

by

of

similarly

nuclei 37

rate

untreated

DPC produced

at

in

elution

from

that

+

was

SSB

produced

SSB produced

indicates

and

nuclei

was

2-Me-g-OH-Et

conditions

(128

produced

produced

whole

The

isolating

SSB

not in

also

in

of

produced

detected

5-ID

breaks

than

concentration-dependent breaks

While

SSB

The DNA

rapidly

process

of

nuclei:

linear.

nucleotides

frequency

the

cell

nearly

more The

per

was

over

and

was

1).

additional

in

L1210

quantification

The

in

isolated nuclei

cells

of

in

level

than

the

break

frequency

failed

to

produce

as

high

28). SSB

in

2C)

whole even

nuclei performed to

cells at

by

concentrations

m-AMSA

without

the

results between

it

and use

obtained the

5-10

drug-induced

of in

could full

not

DNA as

0.33

be

deproteinizing

whole

cells SSB

and

(3-4) DPC.

mM.

detected

Vol. 107, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-Control-Celb

c

0.7

$

0.6

E 5

0.5

4 z!

0.4

Control-Nuclei

2 : 'f

0.3

$ 8 p Y

0.2

E

k IIll III1

0.1

0.0 0.7 0.6 0.5

0.9

FRACTION

%ure

DNA-protein m-AMSA

(0.5

to

2-Me-g-OH-E+ for

(median

of

was

and

3 PM) did

assayed

0.97

crosslinking

SSB and

5-ID m-AMSA

not. DPC

(1

The

1.10

(0.44-1.74).

0.4

0.3

0.2

REMAINING

(Figure

to

10

Theses

isolated

i-1210

PM)

produced

DPC

the of

ratios

nuclei

The

3).

ratio

FILTER

in

5-ID-treated

All

median

ON THE

of DNA from untreated Ll2lD cells (0) irradiated (300 R) cells (A) or nuclei performed using the proteinase-K method 2 ml h-l (see Materials and Methods).

and

7 determinations). 1.58.

I

frequency

and

R-Nuclei

I

OF ‘H-DNA

elution kinetics cell nuclei (0) or 0). Elutions-were at a pump speed of

The

1:

300

I

I

SSB/DPC data

SSB/DPC were

for

5-ID same

by

m-AMSA

nut lei.

concurrently m-AMSA

was

encompassed

(median as

Both

nuclei:

isolated

for

were

the

in

were

ratio

values

cell

or

seen

of in

1 .30

between 9 determinations)

drug-treated

cells

(3-5).

37'C,

Kinetics

of

the

the

SSB produced

DNA effects by

m-AMSA

produced (1

PM)

appeared

579

in

rapidly

isolated in

nuclei: isolated

At nuclei.

Vol. 107, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

C

t

ov I I 1 I I I 1 I 1 I 0

2

4

DRUG

Figure

Over the

half first

drug, minutes.

SSB

2:

6

6

1 o~L..Ll~m--I~-

10

CONCENTRATION

0

20

40

60

60

l/A’ll

The concentration-dependence of intercalator-induced DNA strand break frequency in isolated nuclei (0) or whole cells (0). Nuclei were treated for 30 min at 37°C in nuclei buffer and then diluted 1:20 in iced nuclei buffer. Cells were treated for 30 min at 37°C in RPM1 1630 medium and washed twice in iced medium. Nuclei and cells were then assayed by alkaline elution using the proteinase K method at a pump speed of 8 ml h-l (A) or 2 ml h-l m-AMSA (A); 5-iminodaunorubicin (B); 2-methyl-9-hydroxy(8, Cl. ellipticinium (C). Points are mean + 1 S.D. of at least 3 independent experiments.

of

the

breaks

10 minutes disappeared The

kinetics

observed following

drug

rapidly; of

after

the DPC

a 30 minute treatment

(Figure

half-time

followed

exposure

of

a similar

SSB

4).

occurred After

resealing

pattern

removal was

(Figure

within

10 4).

of

to

20

These

S-IMINODAUNDRUBICIN 300

DNA-PROTEIN

CROSSLINK

FREQUENCY

WWd-equivalents1

Relationship between intercalator-induced DNA single-strand break frequency and DNA-protein crosslink frequency in isolated nuclei. All treatments were for 30 min. SSB and DPC were assayed on aliquots of the same nuclei suspension. Broken lines encompass the area in which SSB frequency and DPC frequency approximate one another within a factor of 2. (A) m-AMSA (0.5 to 3 PM); (B) 5-iminodaunorubicin (1 to 10 PM). 580

the

loo

Vol. 107, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

-0

30 TIME

Figure

data

4:

RESEARCH COMMUNICATIONS

60

FOLLOWING DRUG (minutes1

ADDITION

Kinetics of formation and disappearance of DNA single-strand breaks isolated nuclei exDosed to 1 uM m-AMSA at 37°C. Drum treatment was stopped at 30 min '(arrow) by a 1:30 dilution in nuclei buffer at 37Y. Single-strand break formation and disappearance (0) and DNA-protein crosslink disappearance (A) were studied in 3 independent experiments.

indicate

that

disappearance

the

in

rate

isolated

of

protein-associated

nuclei

is

DNA

identical

to

that

strand-break seen

in

formation

in

whole

and

(3).

cells

DISCUSSION --_ The

ability,

rejoining

in

activity

isolated

stimulated

contlusions.

It

cytoplasmic

metabolism

is

by

the

the

mechanlism

nuc'eus the

and

energy

ent'rely it

:,uggest.s

be

reproduced

DNA is

to

break

from

ATP

in current

enz,lmes

the by

could mammalian

that not

which at

least

an

isolated

is

the

by

some

be topoisomerases nuclei

and

can

to

stimulate

several

this

suggests

produced

is

to

the

localized

It

detergent. to

reseal

breaking

the

the

without

action

present

work.

(2-6).

Topoisomerases

581

that

not

Finally,

seen added

derived

in

cells

can

cofactors.

protein-associated,

intercalator-induced

and

cell

isolation.

effect

to

break

is

and that

suggests

breaks

nuclei

critical

activity

It

intercalator-induced,

can

breaking-

intercalation.

DNA

from

DNA

for

system

derives

the

leads

with

biological

of

cells

removed the

that

the

itself

and

of

reproduce

whole

non-ionic

backbone

response

results

cell

are

probably

rejoining cellular

drug

breaks

DNA

in

necessary

dissociation the

to

m-AMSA

the

hypothesis, and

of

supported

from

resists

breaking part

which

nuclei,

by

suggests

that

Our

cell

of

an

endonuclease DNA

We have

rejoin

distortion,

postulated have

DNA without

which

been

that isolated exogeneous

is such

Vol. 107, No. 2, 1982 energy

(10).

treatment may

The with

indicate

these

detected

treated

Intracellular in

DNA

affinity be that

during

nuclei

breakage its

it

unique

is

may

enzyme,

this

enzyme

during

The initial

present work.

Are

the

the

intercalator

may

the

levels

What

enhance

drug

differs

nuclei,

for

intercalating

this agents

likelihood

in

study

of

no

similarly

indicates

of

required

as

such in

work.

a system

specific The

devoid

a

cells.

If

the

depletion

of

the

raised

by

DNA

breakage?

reproduction

isolation

reproduction of

inability

stimulate can

the

that from

questions

maximally

allow

in

derive

their

the

Finally,

DNA scission?

may

cells,

DNA

m-AMSA

whole

cell

the

depleted

and

for

to

nor

the

2-Me-g-OH-Et

5-fD

precisely

to

possibilities

for

account

whole

nuclei

in

in

a cofactor

intact

also

from

isolated

where

metabolism

present

energy-dependent? in

been

2-Me-g-OH-Et

Other

and

enzyme

be

bring

from

but

could

could

yet,

This

(11-12).

(3-5)

allow

partially effect

the

will

cells

Z-Me-g-OH-E+

the

responsible

expected

factors

responsible of

known

enzyme

isolation

system

processes

substances effects

nuclei

as

breakage

Neither

the

this

whole

not,

of

to

requires

isolated

stimulate

SSB to

required

double-stranded

from

in

buffer.

production

This

solely

gone partially

produce

depleted.

be

drugs

that

case

following

Z-Me-g-OH-Et.

well

break

seen have

nuclei

scission.

is

or

in

be

DNA

nuclei

discernible

inactivate

compound

isolation,

the

readily

could

2-Me-g-OH-Et

other

to

not

in

effects system

in

incubated

produce

this

itself

breaks

did

can

of

could

were

with

likely

RESEARCH COMMUNICATIONS

seen

nuclear

despite

metabolism

which

our

most

that

buffer

potential

compared

in

nuclei

cells

nuclei

form

when

perhaps in

whole the

drugs

is

AND BIOPHYSICAL

DNA breaking

conditions

This

SSB were

that

reduced

that

optimized.

BIOCHEMICAL

of of

the

majority

of

of the cellular

cell

proteins

success.

ACKNOWLEDGEMENTS Thanks to Mrs. Susan Hurst-Calderone for technical assistance Madie Tyler for typing the manuscript. Thanks to Dr. Jan Filipski us his method of nuclei isolation and for access to his data prior publication. Special thanks to Dr. Kurt Kahn for his support of

and to Ms. for teaching to its this work.

REFERENCES 1.

Lown, J.W. 1150-1157.

and

Sim,

S.K.

(1977)

Biochem.

582

8iophys.

Res.

Comm.

II,

this

Vol. 107, No. 2, 1982 2. 3. 4. 5. 6. 7. 8. 9.

.:0. .' 1 . ‘2.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

and Kohn, K.W. (1978) Biochim. Biophys. Ross, W.E., Glaubiger, D.L., Acta 5&, 23-30. Zwelllng, L. A., Michaels, S., Erickson, L.C., Ungerleider, R.S., 20, 6553-6563. Nichols, M., and Kohn, K.W. (1981) Biochemistry Zwelling, L.A., Kerrigan, D., and Michaels, S. (1982) Cancer Res., in press. Zwelling, L.A., Michaels, S., Kerrigan, D., Pommier, Y., and Kohn, K.W. (1982) Biochem. Pharmacol., in press. and Kohn, K.W. (1979) Biochim. Biophys. D.L., Ross, W.E., Glaubiger, Acta 562, 41-50. Zwelling, L.A., Kerrigan, D., Michaels, S., and Kohn, K.W. (1982) Biochem. Pharmacol., in press. Bonner, W.M. (1978) In "The Cell Nucleus" (H. Busch ed.), Vol. Vl, pp. 97-147, Academic Press, New-York. Erickson, L.C., and Zwelling, L.A. (1981) Kohn, K.W., Ewig, R.A.G., A Laboratory Manual of Research Technique" In "DNA Repair: (Friedberg, E.C. and Hanawalt, P.C., eds.) pp. 379-401, Marcel Dekker, New-York. Gellert, M. (1981) Ann. Rev. Biochem. so, 879-910. LePecq, J-B., Dat-Xuong, N., Goose, C., and Paoletti , c. (1974) Proc. Natl. Acad. Sci. USA 7l-, 5078-5082. and Glaubiger, D.L. (1979) In "Mechanism Kohn, K.W., Waring, M.J., of Action of Antieukaryotic and Antiviral Compounds (Hahn, F.E., ed.), p. 195-213, Springer-Verlag, Berlin.

583