Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 28, 1979

Pages 722-729

INTERACTION

BETWEEN OAUNORUBICIN AND CHROMATIN FROM EHRLICH ASCITES TUMOR CELLS

Georgette

Received

Sabeur,

Daniel

Centre

de Biophysique

April

16,1979

Genest

and Genevieve

Moleculaire,

Aubel-Sadron,

F 45045

Orleans

Cedex

SUMMARY Interaction cells

has

been

chromatin sites

of daunorubicin studied

reduced

chromatin

by spectrofluorimetry.

and displays

is

with

at least

ce in the

overall

structure

resistant

to daunorubicin.

Ehrlich

Oaunorubicin

two types

when compared

from

of binding.

acid.

extracted

tumor

interacts

The number

to deoxyribonucleic

of chromatins

ascites

from

with

of binding

There

is

no differen-

cells

sensitive

or

INTRODUCTION The anthracyclin

antibiotic

the

treatment

of acute

leukemias

its

interaction

with

DNA (4,5,6,71

process. merases

In

inhibition

Vitro,

informations

about

polynucleotides teins

action

(12.131

is

available

tance sistant

to daunorubicin

@ 1979

effect

inhibition

to

of DNA synthesis RNA poly-

of a drug-DNA

complex

(8,9,10,111.

However

with

polymers

shows some affinity

of daunorubicin resistance.

at the molecular

by Academic Press. Inc. in any form reserved.

for

other

than

for

pro-

and

t'T41.

level

722

is,

Attempts between

been successful

OOOS-2?lX/79/100722-08$01.00/O of reproduction

agent

has been attributed

enzyme

and daunorubicin

have not

active

such as deoxyribonucleases,

SDS : Sodium dodecyl sulfate PMSF : Phenyl-methyl-sulfonyl-fluoride

Copyright All rights

to the

of daunorubicin

by an encountered

to a difference

activity

upon the

and mucopolysaccharides

reduced

a very

due to the formation

the interaction

are

is

Its

leading

of the drug

The chemotherapeutic strongly

('l,2,31.

of enzymes

and DNA polymerases

not to the direct

daunorubicin

yet.

in some cases, to relate

cells There

this

sensitive is

resisand re-

a decreased

Vol. 88, No. 2, 1979

uptake

of the

cient

to explain

thesis

that

norubicin this

drug

in resistant resistance

cellular

proteins

DNA and chromatin chromatins

(151

could

might

upon the fixation study from

play

a role

cannot

step

Ehrlich

ascites from

with

the

tumor

ascites

suffi-

The hypo-

be ruled

of dau-

out.

From

the influence

of

to DNA in chromatin. fixation

cells

tumor

is not

factors.

considered

of daunorubicin

concerned

diminution

in the mode of action

of resistance

is

extracted

but this

be due to several

we have in a preliminary

The present

between

which

proteins

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cells

and (or-1 the development

standpoint,

nuclear

BIOCHEMICAL

of daunorubicin

as well

cells

as the

sensitive

to

comparison

or resistant

to the drug. MATERIALS

AND METHODS

Daunorubicin is a generous gift from Rhane-Poulenc, S.A., France. Solutions of daunorubicin were kept in the dark for one week at most. Concentration of daunorubicin was calculated using an extinction coefficient of 10,000 M-1 at 460 nm. Ehrlich ascites tumor cells : CD 1 female mice (Charles River, France] 6-8 weeks old (20 gl were inoculated intraperitoneallv with IO6 cells. Ascites fluid was collected 8 days after inoculation and either used within the hour (chromatin preparation] or washed with phosphate-NaCl buffer and then frozen (DNA preparation]. Resistance was developed by treatment with subtherapeutic doses of daunorubicin [0.X mg/l kg1 given intraperitoneally for 5 days. Ascites fluid was collected 2 weeks after inoculation of resistant cells. Ascites chromatin was prepared according to Paoletti (161. In this method, cells were washed (5 mn, 1,000 gl once with sucrose 0.25 M, EOTA 5 mM, pH 8 and four times with sucrose 0.25 M, EDTA 0.1 mfl, pH 8. During this washing each run, pH was adjusted to 6 by addition of NaOH 20 mM. Chromatin step, after was then extracted as described by No11 et al. (171. Micrococcal nuclease [EC 3.1.4.7.1 digestion was performed during 10 mn at 37' [ZO units/ml). Our chromatin preparations contained about 70 % of proteins, 22 % of DNA and 6 % of RNA as determined according to ref. 16, 19, 20 respectively. Concentration of chromatin was calculated assuming an extinction coefficient of 7,300 ~-1 cm-1 at 260 nm. DNA preparation : Ascites DNA was prepared from purified nuclei according to Barthelemy-Clavey et al. (101. Concentration of DNA was calculated using an extinction coefficient of 6,600 ~-1 cm-1 at 260 nm. Electrophoresis pH 7.6, B-mercaptoethanol according to Weintraub

: Chromatin was lyopholized, dissolved in Tris 0.02 M, 5 %, SDS 2 % and submitted to electrophoresis in SDS et al. [Z'll. Proteins were stained with coomassie blue.

Binding experiments : Addition of nucleic acid (41 or chromatin to a solution of daunorubicin results in a decrease in the fluorescence and absorption intensities of daunorubicin. This decrease is dependent on the concentration of added nucleic acid or chromatin and can be used to determine the amount of bound dye.

723

BIOCHEMICAL

Vol. 88, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1) Spectrofluorimetric titrations were carried out with a Jobin-Yvon spectrofluorimeter modified in the laboratory. All the experiments were performed at room temperature in Tris-HCl 0.01 M, NaCl 0.01 M, EDTA 0.2 mM. pH 7.5, buffer. Chromatin and DNA were dialyzed against this buffer. Each intensity reading was corrected by an appropriate blank with DNA, chromatin or buffer. Oaunorubicin was excited at 485 nm and fluorescence emission was measured at 570 nm. : Absorption 21 Absorption Beckman Acta III spectrophotometer. procedure similar to that described was measured at 480 nm.

spectra were recorded on a Cary 15 or a Binding experiments were performed by a by Mijller and Crothers (221. Absorbance

Binding isotherms were determined at constant polymer concentration (1.5-3 x 10-5 M, expressed in nucleotide), the concentration of added daunorubicin varied between 5 x 10e7 and 1.5 x 10m5 M for DNA and between 1 x IO-7 and 5 x IO-5 for chromatin. Data were analyzed using Scatchard's relationship: r/c = K [n-r) where r is the dye bound per DNA or chromatin phosphorus, c is the concentration of free dye, n and K are respectively the apparent number of binding sites and the apparent binding constant, both determined from a plot of r/c versus r.

RESULTS AND DISCUSSION Protein ted in fig. histone

from grity circular

tin.

An isobestic

tide/dye

ratio

in fig.

for

the

free

intensity

amounts

4a.

with

that,

so far,

by 'cryptic

represen-

number from

or five

of non

resistant

preparations

to proteolysis four

is

a great

chromatin

of chroma-

than

preparations

days.

The inte-

digestion

complexation

and a decrease

point

Scatchard's sented

been shown that

2, one can see that

(fig.

with

cells

[21,23)

and by

(24,251.

Fluorescence of increasing

sensitive

along

is obtained

has been checked

in a red shift In fig.

present

and have to be used within

has already

(6).

from

are more susceptible

chromatins

dichroism It

are

has to be noticed

cells cells

of both

results

it

resistant

sensitive

histones

chromatin

The same pattern

Nevertheless, from

of ascites

1. The five

proteins.

cells. tin

content

in intensity

dye is

of daunorubicin and almost

to DNA

of the absorption

same phenomenon

and bound

of chromatin

of daunorubicin

is

found

is

with

chroma-

at 540 nm.

strongly

totally

observed

maximum

reduced

quenched

by addition

at high

nucleo-

31. plots

obtained

The curvature

from

fluorescence

of the isotherm

724

measurements indicates

that

are ascites

reprechro-

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

H2aH2b

Fig.

1 - Densitometric from sensitive

matin

displays

assumes

at

that

the

cooperativity, to 0.08

[fig.

in

are high is

ascites

binding.

sites

carried

a slight

proteins

of

approximation

if

cooperativity

without

affinity only

electrophoresis

a first

In

independent,

chromatin

obtained

ding

sites engaged

with on

decreases

change

in

and

K = 7 x

out

by

spectrophotometry

be

in

are

in

good

agreement

calf

thymus

and

interaction

when

one

on

ethidium

expected

from

We can

assume

chromatin

is

in

DNA

daunorubicin

daunorubicin

could

(27,281.

is

There

studies

tion

it

of

of

gel

IO6

from the

M

-1

0.17

binding

for

one

or antifor

DNA

constant

ascites

confirm

:

DNA. these

re-

4al.

fluorimetric

DNA

number

polyacrylamide chromatin.

modes

sites

experiments

Results

of

binding

for

T”l-’

Complementary

observed

two

chromatin.

K = 5 x IO6

sults

least

the

for

scans of ascites

a chain

studies that

the

explained of

reduced by

a less

nucleosomes.

725

deals

with DNA

with

previous

[261.

The

chromatin

of

accessibility

difference instead

bromide-chromatin

number

spectro-

interac-

intercalating of

dye its

bin-

DNA when

Vol. 88, No. 2, 1979

4i)o

BIOCHEMICAL

500

AND BIOPHYSICAL

SPO

RESEARCH COMMUNICATIONS

540

Fig.

2 - Visible absorption spectra 1. Free daunorubicin 2. Chromatin 1.5x10-4 M 3. Chromatin 5.3x10-4 M.

Fig.

3 - Decrease of daunorubicin fluorescence chromatin and DNA. A DNA l Chromatin C = nucleotide concentration Daunorubicin concentration : 10m7 PI.

580

of daunorubicin-chromatin

726

intensity

complexes.

upon addition

of

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

f 10-Q

to

I I

b Fig.

4 - Scatchard's DNA. al Ascites tion. Ascites bl Ascites

If pared

from

sensitive sites

One can argue binding

that constant

cannot

distinguish

matin.

Fluorescence

chromatins

confirm

for

chromatin

the

binding

from

the

of daunorubicin

sensitive

DNA : 0 fluorescence, chromatin from resistant

one compares

ber of binding

the

plots

0.1

behaviour

and resistant

cells

of daunorubicin cells,

as the

Scatchard's

plots

can only

and the number

of binding

small

polarization the

similarity

differences studies observed

to be published).

727

towards

of the binding give

4b1 that constant

an order

at the

nucleosome bromide

daunorubicin

pre-

the num-

are the

of magnitude

and that

with

A absorp-

chromatins

sites

of ethidium

and

fluorescence.

one can see (fig.

as well

between

value

to chromatin

: A fluorescence,

o absorption. cells : x

.r

0.2

same. for

our experiments level with

in chro-

the

CO. Genest

two et al.

Vol. 88, No. 2, 1979

There nucleosome

is

amount

daunorubicin is

the

binding.

it

full

This for

seems worth

preserve

action

is

to the

drug.

upon the the

were

result

noticing

that

like

chromatin

Oaunorubicin

binding

which

extracted

daunorubicin

action

is

in progress

on the

resistant

from

proteins.

in

nating

is

well

of non-histone

pro-

like

structure.

H, and H3

known to interact

some non-histone

This

proteins

complex.

cells

to DNA in chromatin

of non-histone

subcellular

of in

of histones

in a nucleoprotein

not participate

and on the

by proteolysis

of a polynucleosome

daunorubicin

that

tins

of resistant

has been observed

the bulk

digests

of the

structure.

with

work

that

are part

a preparation

but depleted

limit

hypothesis

Presently,

proteins

: no difference

suggests

DNA engaged

presence

with

of histones

with

observed

performed

the maintenance

We have shown that also

some non-histone

proteins

a nucleosome

DNA interacts

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

set

of non-histone

not required

Besides,

evidence

Experiments

containing

a great

still

recent

(29,301.

chromatin

teins

8lOCHEMlCAL

localization

The same inter-

sensitive

or resistant

does not

seem to depend

observation or other

composition

of the proteolytic

does

cellular

or in the phenomenon proteic

with

not rule proteins

out do

of resistance. of the activity

two chromacontami-

chromatin.

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