Action of neocarzinostatin on cell nuclei: Release of specific chromatin

Vol. 104, No.

3, 1982

Februory

1982

11,

ACTION

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages

OF NEOCARZINOSTATIN

ON CELL

Takumi Department

Received

AND BIOPHYSICAL

Hatayama

and

of Biochemistry, l-4-54, Asahi-machi,

December

21,

NUCLEI:

RELEASE

Munehiko

Osaka City Abeno-ku,

OF SPECIFIC

B89-896

CHROMATIK

Yukioka. University Osaka

545

Medical Japan

School,

1981

Neocarzinostatin solubilized chromatin from rat liver nuclei in the presence of Z-mercaptoethanol with little increase in acid-soluble materials. When DNA from the chromatins was electrophoresed on neutral and alkaline agarose gels, a series of bands with a multiple of a monomeric nucleosomal unit was observed, and the ratio of double-strand to single-strand breaks DNA in chromatin was high (1:7) as compared with findings in the case of purified DNA (1:30). Hybridization analysis of the DNAs with cDNA of liver polysomal poly(A)+mRNA revealed that neocarzinostatin solubilized a specific chromatin and which differed from the active chromatin which was preferentially excised by micrococcal nuclease.

of

INTRODUCTION

The of

antitumor

Streptomyces

molecular

DNA

causes

DNA

sulfhydryl

is

of

the

from

scission

activities

mechanism

10

the

intact

chromatin

be

considered. In

--in

drug

to

of

cell

the

of

of

liver.

is

induce

DNA

strand

in

HeLa

and

unknown.

work,

Abbreviations: complementary of hybridization

which

we

solubilized

possesses

found

binds

precise elucidate

the

cellular

that

NCS

specific

to

and

inhibition

its

A non-protein

cytotoxic

and by for

the

NCS

DNA

is

produced chromatin

accumulated

appears

DNA

-~in

an

ref.

exist

NCS

31. by

of

the DNA

chromophore vitro

DNA strand

intercalative

action

in

organized

DNA damage intact

cells,

[12,13]

has

to

double-strand

fragments

NCS, neocarzinostatin; poly(A)+mRNA, DNA; Cot, concentrations of DNA (mol (set); RF DNA, replicative form of

that

between

NCS-induced

high

a

stimulated

[5].

to

a filtrate

with

in

greatly

mechanism

To

has

[reviewed

breakage

directly the

action a reaction

cells

from

polypeptide

evidence

A correlation

[6],

isolated

single

its in

[4].

in

DNA and

for and

vitro

[7-91

present the

target in vivo -__

However,

(NCS), acidic

Considerable

growth

structure

an

[1,2].

antibiotic

cells

the

breaks

700

important

[lO,ll].

the

is

breakage

and

isolated

cDNA, time

an

strand

replication

rat

of

compounds

ability

neocarzinostatin

carzinostaticus,

weight

cellular

in

antibiotic

from

the

polyadenylated nucleotides/l) DNA.

nuclei

mRNA; times

0006-291X/82/030889-08$01.00/0 889

Cop.vright 0 I982 b.v Academic Press, Inc. All rights of reproduction in anv ,form reserved.

Vol. 104, No. 3, 1982 MATERIALS

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AND METHODS

NCS is

a product

of Kayaku Antibiotics

Research

Co.,

Tokyo,

Japan.

Preparation of nuclei Rat liver nuclei were prepared using the procedures of Tata and Baker [14], except for slight modifications as follows: the nuclei recovered through a 2.3 M sucrose layer were washed twice with 0.25 M sucrose-l mM MgC12 without triton X-100. NCS digestion of nuclei The purified nuclei were suspended in a medium containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.2 mM phenylmethylsulphonyl fluoride and 8% glycerol (digestion buffer) at a concentration of 40 A260. The nuclear suspension was incubated in the dark with NCS in the presence of 10 mM 2-mercaptoethanol at 30°C for the indicated time. The reaction was terminated by chilling the tube on ice and the nuclei were lyzed by addition of 0.1 M EDTA (pH 7.0) to 1 mM. After centrifugation at 10 000 xg for 10 min at 4"C, the recovered supernatant and pellet fractions were designated as S and P, respectively. Purification of DNA DNA was extracted and purified from undioested nuclei or fractionated chromatin, by digestion with proteinase K, phenol-chloroform extraction and RNase treatment as described previously [15]. In case of the S fraction, the chromatin was ethanol-precipitated in the presence of 0.2 M Na acetate (pH 7.5) and DNA was extracted as above. Electrophoretic analysis of DNA Native DNA sample was electrophoresed on a 1.85% agarose slab gel in 40 mM Tris, 30 mM Na acetate and 2 mM EDTA (pH 7.8, adjusted by acetic acid), as described previously [15]. Denatured DNA samples were analyzed on a 1.85% agarose slab gel in 30 mM NaOH and 2 mM EDTA, as described by McDonell --et al. [16]. The gels were stained with 20 pg/ml ethidium bromide in 5 mM EDTA (pH 7.0), transilluminated with ultraviolet light and photographed through a red filter. Densitometry was performed using a Shimadzu TLC scanner (CS-900) and a mean strand length of DNA fragments was evaluated from the densitometric pattern. Synthesis of complementary DNA to rat liver mRNA Rat liver polysomal RNA was prepared by the method described by Sippel et al. [17] and poly(A)+mRNA was isolated from the polysomal RNA by si?jo(dT)-cellulose chromatography. [3H]-labeled single-stranded DNA complementary to the mRNA was synthesized using [3H]dCTP (18.4 Ci/mmol, Amersham), according to the method described by Kameji -et P. al. [18]. The length of the cDNA was between 300-2 000 nucleotides and the specific activity was approximately 1x107 cpm/pg. DNA-cDNA hybridization DNA prepared from undigested nuclei or fractionated chromatin was sheared by sonication using several-intermittent 20 set bursts, to a mean singlestrand length of 400-500 nucleotides, as determined by alkaline agarose gel electrophoresis. Hybridization of [3~]~~~A (10 rig/ml) to these DNAs (10 mg/ml) was carried out in 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.6 M NaCl and 0.01% sodium dodecyl sulphate under paraffin oil. The mixture were treated at 100°C for At each time, a 20 ul aliquot 5 min and incubated for varying times at 68°C. was withdrawn into 1 ml of a medium containing 30 mM Na acetate (pH 4.5), 0.3 M NaCl, 3 mM ZnSO4 and 10 pg/ml of denatured and sonicated calf thymus DNA, and divided into two equal portions. Sl nuclease (1 000 units) was added to each one of two portions and both portions were incubated at 45°C for 2h. After addition of sonicated calf thymus DNA (100 pg) as a carrier, 10% trichloroacetic acid-precipitable radioactivity was measured. Sl nuclease890

Vol. 104, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

50 (A)

t

lo

7.u 33 40 50 60 Time

I

(mid

50

(B)

t

10’ +*--* 0

____-

0

-0

e-e---------

200

Loo

c em

600

NCS Concentration

(pg/ml)

Release of chromatin from rat liver nuclei by neocarzinostatin. (A) Fig. 1. Time course: rat liver nuclei were incubated with or without NCS (200 pg/ml) in the presence or absence of 2-mercaptoethanol at 30°C. At the indicated time, aliquots of the nuclear suspension were withdrawn and fractionated as described in Materials and Methods. The digestion was monitored by the % A260 released into S () and by the % A260 rendered acid-soluble (-----). 0, + NCS + 2-mercaptoethanol;A, + NCS - 2-mercaptoethanol;O, - NCS + 2-mercaptoethanol; - NCS - 2-mercaptoethanol. (B) NCS dependency: rat liver nuclei were 0, incubated with various concentrations of NCS in the presence of 2-mercaptoethanol at 30°C for 30 min; the % A260 released into S (M) and the % A260 rendered acid-soluble (O--G) were analyzed.

resistant fraction were corrected to

RESULTS Action

AND of

To

isolated the

action

at

with

or

digestion

(Fig. 15 min

30°C

(S) lA), and

2-mercaptoethanol. acid-soluble 2-mercaptoethanol,

subtracted concentration

from

the (0.18

data. N Naf)

Cot values [19-j.

nuclei

determine

supernatant

initial

cDNA (2.0%) was standard cation

DISCUSSION

NCS on

incubated into

of the

of

and

pellet

the

soluble

gradually

were much

smaller

chromatin,

NCS, (P)

and

to

even

rat

the

As (S)

60 min after

released.

in

amounts

of

891

control the

seen

presence digestion, samples soluble

nuclei

digest

increased the

60 min In

liver

nuclear

fractions.

chromatin up

However, materials

NCS on

without

in

were

was the

time

rapidly of

fractionated course

during NCS

of the

and

no more

than

without

NCS or/and

chromatin

were

6.0%

of

Vol. 104, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(A>

03

123456

Fig. 2. Electrophoretic analysis of DNA from NCS-treated nuclei. Rat liver nuclei were incubated with various concentrations of NCS in the oresence of E-mercaptoethanol at 30°C for 1 h and fractionated into S and P.' DNA was purified from the fractions and analyzed by neutral (A) or alkaline (B) agarose gel electrophoreses, as described in Materials and Methods. DNA from the S fraction only is shown. Lane No. 1, no NCS; 2, 100 pg/ml; 3, 200 C(g/ml; 4, 400 ug/ml; 5, 800 pg/ml; 6, ADNA-Hind III digest. The numbers between (A) and (B) represent length in nucleotides, calibrated by *DNA-Hind III and 9x174 RF DNA-Hae III digests (Biolabs).

obtained

during

increased

to

during

the

soluble

60 4.5%,

60

min

the

of

were

with

NCS

gel

the DNA

The

lengths 400

of and

kilobase

DNA the

pg/ml,

pairs,

the

was is

30%

Thus,

chromatin

and

1.5%,

and

NCS

in

the

dose (Fig.

presence

manner

the

increased, materials

in

but

acid-

respectively.

of was

acid-soluble

nuclei,

dose-dependent

a series observed,

nuclei of

to

with

the

restriction

bands

with

1B).

of

with

little

drug,

increase

of

DNA

the

chromatin

(S)

solubilized

estimated

as

approximately

2A).

892

Under

analyzed

fragments,

10, alkaline

agarose

of

linker as

DNA between

reported

by

others

NCS concentrations. mean

with

by

a multiple

that

the

were

(Fig.

were

indicating

susceptible

decreased

marker

respectively

[12].

soluble

chromatin of

the

nuclease the

concentrations

soluble

NCS-treated

2A,B),

unit

of

various

materials

materials.

particles

DNA from

800

and

from (Fig.

from

about

increase

acid-soluble

endogenous

were

from

a time

the

of

with

acid-soluble

size

Calibrating

of

pg/ml)

chromatin

fragments

core

which

increases

the

nucleosomal

nucleosomal [20].

(200

a concomitant

electrophoresis

monomeric

action net

incubated

in

in When

the

NCS

solubilized

2-mercaptoethanol, increase

to

in

2-mercaptoethanol,

dependently, Thus,

due

by

nuclei

presence

incubation,

incubation,

materials

When

min

double-strand

NCS at 5.5,

0, 4.0,

conditions,

100, 3.0

200, and much

1.8

Vol.

BIOCHEMICAL

104, No. 3. 1982

AND BIOPHYSICAL

Equivalent

RESEARCH COMMUNICATIONS

Cot

DNA-cDNA hybridization. (A) Rat liver nuclei were incubated with of NCS in the presence of 'Z-mercaptoethanol at 30°C for 8 (0,O) or 30 min (a,A). DNA was isolated from S (O,A), P (.,A) or undigested nuclei (Cl), and hybridized with [3H]cDNA of liver polysomal poly(A)+mRNA, as described in Materials and Methods. (B) The nuclear suspension (100 A260) in the digestion buffer containing 0.25 ti CaC12, was incubated with 20 units/ml of micrococcal nuclease (Worthington) at 30°C for 8 min (4% acidsolubility). The reaction was terminated by chilling the tube on ice and by addition of EGTA to 0.5 n+l. The chromatin was separated into supernatant (S,O) and pellet fractions. The pellet was extracted with a medium containing 10 mM Tris-HCl (pH 8.0), 8% glycerol, 1 mM dithiothreitol and 1 mM EDTA, and centrifuged (20 000 xg, 10 min) to obtain the extractable (PS,A) and residual (P,O) fracti ns. DNA from the fraction or undigested nuclei (Cl) was hybridized with [ 3 H]cDNA as above. In the digestion, 22, 48 and 30% were recovered in S, PS and P fractions, respectively. Of A260 F 00 pg/ml

smaller

sizes

lengths

were

of

the

about

of

0,

residual

1.5

DNA were

approximately

concentrations from

the

100,

10,

2.5,

200,

400

chromatin

(1.2-1.8)

corresponding

seen,

(P)

times

doses

of

longer NCS,

in

shown).

The

ratio

of

double-strand

the

was

approximately

1:7.

data

DNA was were breaks

digested

observed ratio

with (data was

was

1.0

2B, and

than

those

of

the

same

and

the

reported

the

and

single-strand

kilobases,

DNA soluble

when no to [Zl].

DNA were

chromatin

(S)

conditions

double-strand previously

NCS

lengths

breaks, hand,

conditions,

at

When

strand

alkaline

single-strand other

mean

respectively.

mean

neutral

and 0.65

pg/ml,

analyzed,

to

as

Fig.

800

On the

shown), 1:30,

in

1.5, and

both

NCS under not

about

as

calculated purified

discrete single-strand

at

(data

rat

not from

liver

ladder-bands

Vol. 104, No. 3, 1982

BIOCHEMICAL

NCS produces lesser

extent,

[22,23].

single-strand

linker

region

In this

random

context,

NCS-treated

placement it

nuclei,

were

treated

obtained

from

were

As shown

with

hybridized

in Fig.

38,

nuclease-digested

with the

chromatin

(S) from

the level

of the

of subsets

The DNA of soluble

chromatin

extent

in the

coccal

nuclease-digested

exhibited

sequences

almost

Interestingly,

nuclei,

the

supernatant

fraction

was not

of 1 mM EDTA. In the case of the soluble

chromatins

chromatin the

(P)

DNA from

appearing

nuclei

nuclei

sequences. recognize

These the

particular transcribed

results

same features

was depleted nuclei.

were

nuclei

DNA.

slightly

or without situation

while enriched

was depleted that

of the chromatin

in the addition

of the

the DNA of residual

the same as the total chromatin

in some subsets

NCS and micrococcal structure

DNA,

in the sequences

the DNA of residual

demonstrate

(PS)

recovered

a reciprocal

3A). to some

chromatin

with

at two

In micro-

of chromatin

was almost

whereas

(Fig.

as the total

different

DNA

to be saturated

the DNA has only

nuclei

In NCS-digestion,

8 min-treated

nuclease-digested

that

the

kinetics

chromatins,

was observed.

in the

the amount

significantly

residual

30 min-treated

was found

the DNA of EDTA-extracted

in the poly(A)+mRNA;

micrococcal

breaks

from

poly(A)+mRNA.

In contrast,

from 8 min-treated

nuclei,

rat

untreated

in the poly(A)+mRNA

30 min-treated

same hybridization

in NCS-treated

cell

sequences.

appearing

[24,25].

nuclei

appearing

was that

the

(S) from micrococcal

DNA, indicating

from

than

for

and

nuclease,

polysomal

sequences

by others

of the sequences

breaks

as DNA from

liver

chromatin

NCS-treated total

of double-strand DNA damage of

and DNAs of the chromatin

in the

reported

of soluble

ratio

that

DNA.

on transcribing

as well

of rat

was enriched

as previously

nuclease

over

purified

of double-strand

agents

[3~]~~~~

two thirds thirds

a high in cellular

nuclei,

the DNA of soluble

nuclei

poly(A)+mRNA,

these

with

in the

of NCS and micrococcal

treated

in DNA

increased

seemed to be responsible

micrococcal

to a

of producing

probably

breaks

production

the effects

feasibility

is

that

in the cells,

to compare

nuclei

fractions

the

and,

of DNA in the chromatin,

to NCS, the

has been found

and that

non-repairable

In order

portion

emphasizing

(1:5)

[21]. of NCS as compared

liver

a limited

acid

members of base pair,

of single-strand

is worth

breaks

RESEARCH COMMUNICATIONS

at deoxythymidylic residues,

of DNA in the chromatin

HeLa cells

were

killing Effect

as only

breaks

to single-strand which

acid

of DNA, is accessible

double-strand due to the

breaks

at deoxyadenylic

In addition,

AND BIOPHYSICAL

and that

(P) from of the

nuclease

do not

NCS attacks

a

portion of chromatin, the DNA of which is depleted in the sequences. Thus NCS seems to preferentially introduce strand

in chromatin

which

differs

from

transcriptionally

This might be related to the specific inhibition without an effect on RNA synthesis in the intact 894

active

of DNA replication cells [26].

chromatin. by NCS, but

Vol. 104, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

The --in vitro action of NCS on the chromatin described here seems to be closely related to its action detected in vivo [3]. Therefore, it is likely -__ that chromatin structure plays a critical role in the action of NCS in vivo. As NCS recognizes the active nuclei,

chromatin

the chromatin which

NCS can be effectively

is

structure sensitive

which

specifically

to micrococcal

utilized

for

studies

differs

nuclease on the specific

from

in the cell chromatin

structure.

ACKNOWLEDGEMENTS We thank Professor N. Ishida (Tohoku University) NCS, Dr. M. Obinata (the University of Tokyo) for the transcriptase, and Miss M. Ohara (Kyushu University) the manuscript. This study was supported in part by Ministry of Education, Science and Culture of Japan.

for the generous gift of gift of AMV reverse for critical reading of a Grant-in-Aid from the

REFERENCES 1. 2. 3.

4. 5. 6. 7. ;: 10. 11. 12. 13. 1:: 16. 17. 18. 19. 20.

Ishida, N., Miyazaki, K., Kumagai, K. and Rikimaru, M. (1965) 3. Antibiot. 18, 68-76. Meienhofer, J., Maeda, M., Glaser, C.B., Czombos, J. and Kuromizu, K. (1972) Science 178, 875-876. Goldberg, I.H., Hatayama, T., Kappen, L.S., Napier, M.A. and Povirk, L.F. (1981) Molecular Actions and Targets for Cancer Chemotherapeutic Agents, Bristol-Myers Cancer Symposium in Cancer Research (Sartorelli, A.C., Laxo, J.S., & Bertino, J.R., Eds) pp 163-191, Academic Press, New York. Beerman, T.A. and Goldberg, I.H. (1974) Biochem. Biophys. Res. Commun. 59, 1254-1261. Beerman, T.A. and Goldberg, I.H. (1977) Biochim. Biophys. Acta 475, 281-293. Napier, M.A., Holmquist, B., Strydom, D.J. and Goldberg, I.H. (1979) Biochem. Biophys. Res. Commun. 89, 635-642. Kappen, L.S., Napier, M.A. and Goldberg, I.H. (1980) Proc. Natl. Acad. Sci. USA 77, 1970-1974. Ohtsuki, K. and Ishida, N. (1980) J. Antibiot. 33, 744-750. Suzuki, H.,Miura, K., Kumada, Y., Takeuchi, T. and Tanaka, N. (1980) Biochem. Biophys. Res. Commun. 94, 255-261. Povirk, L.F. and Goldberg, I.H. (1980) Biochemistry 19, 4773-4780. Povirk, L.G., Dattagupta, N., Warf, B.C. and Goldberg, I.H. (1981) Biochemistry 20, 4007-4014. Hewish, D.R. and Burgoyne, L.A. (1973) Biochem. Biophys. Res. Commun. 52, 504-510. Noll, M., Thomas, J.O. and Kornberg, R.D. (1975) Science 187, 1203-1206. Tata, J.R. and Baker, B. (1978) J. Mol. Biol. 118, 249-272. M. (1981) Biochim. Hatayama, T., Omori, K., Inoue, A. and Yukioka, Biophys. Acta 652, 245-255. McDonell, M.W., Simon, M.N. and Studier, F.W. (1977) J. Mol. Biol. 110, 119-146. Sippel, A.E., Hynes, N., Groner, B. and Schiitz, G. (1977) Eur. J. Biochem. 77, 141-151. Kameji, R., Obinata, M., Natori, Y. and Ikawa, Y. (1977) J. Biochem. 81, 1901-1910. Britten, R., Graham, D.E. and Neufeld, B.R. (1974) Methods in Enzymology (Grossman, L. & Moldave, K. Eds) Vol. 29, pp 363-418, Academic Press, New York. Kuo, M.T. and Samy, T.S.A. (1978) Biochim. Biophys. Acta 518, 186-190. 895

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BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Hatayama, T. and Goldberg, I.H. (1979) Biochim. Biophys. Acta 563, 59-71. Hatayama, T., Goldberg, I.H., Takeshita, M. and Grollman, A.P. (1978) Proc. Natl. Acad. Sci. USA 75, 3603-3607. D'Andrea, A.D. and Haseltine, W.A. (1978) Proc. Natl. Acad. Sci. USA 75, 3608-3612. Bloom, K.S. and Anderson, J.N. (1978 ‘) Cell 15, 141-150. Bellard, M., Gannon, F. and Chambon, P. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 779-791. Homma, M., Koide, T., Saito-Koide, T , Kamo, I., Seto, M., Kumagai, K. and Ishida, N. (1970) Progress in An imicrobial and Anticancer Congress of Chemotherapy, Preceedings of the 6th International Chemotherapy, Vol. 2, pp 410-415, Un versity Park Press, Baltimore.

896