Biochimica et Biophysica Acta, 434 (1976) 509--512

© ElsevierScientific Publishing Company, Amsterdam-- Printed in The Netherlands

BBA Report BBA 31205 BINDING OF DAUNOMYCIN TO NONHISTONE PROTEINS FROM RAT LIVER

HIDEAKI KIKUCHI and SHOJIRO S A T O

Research Institutefor Tuberculosis, Leprosy and Cancer, Tohoku University,Sendal-980 (Japan) (Received February 5th, 1976)

Summary Evidence for binding of daunomycin to nuclear nonhistone proteins from rat liver was obtained by equilibrium dialysis. This binding increased in the presence of 8% glycerol (v/v) and was specifically high for nonhistone proteins compared with other proteins.

Daunomycin, an antitumor antibiotic agent, is believed to bind firmly to DNA and thereby to inhibit DNA and RNA synthesis [1--3]. During studies on the interaction of daunomycin with chromatin from rat liver, we noticed the possibility that daunomycin binds to nonhistone proteins [4]. Although many accounts have been published on the binding of daunomycin to DNA [1, 4--7], there seems to be no report about the interaction of this antibiotic with nuclear proteins. The present paper aims at confirmation of this binding by equilibrium dialysis. Liver nuclei from adult Donryu rats (about 120 g) were prepared by the method of Teng et al. [8]. From the purified nuclei, chromatin was prepared using the procedure of Huang and Huang [9]. The chromatin was suspended in distilled water and this made up to 3 M NaC1/0.01 M Tris, HC1 (pH 7.9) by adding solid NaC1 and 1 M Tris. HC1 (pH 7.9). After stirring in the cold for 60 min, the salt-dissociated chromatin was applied to a Bio-Gel A-50 m column (4.0 × 90 cm). The protein pool was dialyzed at 4°C twice for 5 h and then once for 12 h against 50 vol. of 7 M urea/0.01 M sodium acetate buffer (pH 5.2)/0.23 M NaC1 and fractionated on an SP-Sephadex column (1.5 × 30 cm) according to the method of Graziano and Huang [10]. The non-adsorbed fraction was used as nonhistone proteins. Dialysis tubing (8/32, Visking Company) containing 10--180 pg nonhistone proteins (1.0 ml) in 0.01 M Tris. HC1 (pH 7.5)/0.05 M KC1 was dialyzed at 4°C for 20 h against 100 ml of the same buffer-salt solution

510 containing 1 pM daunomycin (kindly donated by Meiji Seika Co., Ltd., Tokyo). After dialysis, aliquots of the inner (protein solution) and outer solutions were withdrawn and mixed with 3 vol. of n-butanol respectively in screw-capped test tubes. The mixture was vigorously shaken and centrifuged at 900 × g for 5 min. Two ml of the organic phase were removed for measurem e n t of fluorescence intensity with an Hitachi MPF 2-A fluorescence spectrophotometer [11]. The light emission was determined at 582 n m when the sample was excited at 467 nm. Values for binding of daunomycin were calculated using the calibration curve, which was prepared by treating the known concentrations of daunomycin in 0.01 M Tris. HC1 (pH 7.5) in the same manner. Fig. 1 shows that the amount of daunomycin bound to nonhistone proteins increased with increasing concentrations of protein until a m a x i m u m was reached, at which point the binding levelled off. This behavior may be due to aggregation of nuclear nonhistone proteins occuring at high concentrations [12]. A slight precipitate of nonhistone proteins which formed on dialysis was indeed observed at concentrations where levelling off was observed In an attempt to prevent the aggregation of nonhistone proteins and to increase the daunomycin-binding activity, various reagents were added to the dialysis buffer. Fig. 2 shows that 8% glycerol (v/v) increased the binding about 2 fold. Therefore, subsequent experiments were performed in the presence of 8% glycerol (v/v). Since the marked interaction between daunomycin and DNA is well known [1, 5--7], which we confirmed in the previous work [4], nonhistone proteins used were tested for possible contamination by DNA. DNA could 0.3

~0.2

0.4 o cE 0.3

c

g~'0.2

o~

0

~ 0.1~

0

.~o

~k~

1~

Nonhistone proteins (jug/ml)

~oo

0i 0

5 10 Glycerol % ( V / V )

15

Fig. 1. R e l a t i o n s h i p b e t w e e n c o n c e n t r a t i o n o f n o n h i s t o n e p r o t e i n s a n d a m o u n t o f b o u n d d a u n o m y c i n . One*m/solutions cont~!ning various amounts of nonhistone proteins in 0.01 M Trls*HCl (~H 7.5) and 0 . 0 5 M KC] w e r e d i a l y z e d a g a i n s t 1 0 0 m l o f t h e s a m e m e d i u m p l u s 1 # M d a u n o m y c i n a t 4 C f o r 2 0 h . A f t e r d i a l y s i s , e q u a l v o l u m e s o f i n n e r a n d o u t e r s o l u t i o n w e r e e x t r a c t e d w i t h 3 voL o f n - b u t a n o l a n d t h e a m o u n t o f d a u n o m y c i n w a s d e t e r m i n e d as d e s c z i b e d i n t h e t e x t . Fig. 2. E f f e c t o f v a r y i n g g l y c e r o l c o n c e n t r a t i o n o n d a u n o m y c i n b i n d i n g t o n o n h i s t o n e p r o t e i n s . O n e - m / s o l u t i o n s c o n s i s t i n g o f n o n h t s t o n e p r o t e i n s ( 8 6 . 4 # g ) ~ 0 . 0 1 M T r i s , H C l ( p H 7 . 5 ) , 0 . 0 5 M KC1 a n d v a r y i n g c o n c e n t r a t i o n s o f g l y c e r o l w e r e d i a l y z e d a t 4 C f o r 2 0 h a g a i n s t 1 0 0 voL o f t h e s a m e r e s p e c t i v e medium plus 1 #M daunomycin. After dialysis, equal volumes of inner and outer solution were e x t r a c t e d w i t h 3 vol. o f n - b u t a n o l a n d d a u n o m y c i n w a s f l u o r i m e t r i e a n y d e t e r m i n e d . T h e a m o u n t o f d a u n o m y e i n b o u n d t o n o n h i s t o n e p r o t e i n s is p l o t t e d a g a i n s t g l y c e r o l c o n c e n t r a t i o n (v/v).

51]

TABLE I EFFECTS OF ENZYMATIC TREATMENTS

OF NONHISTONE PROTEINS ON BINDING ACTIVITY

One-ml mixtures of nonhistone proteins and DNAase or pronase in 0.01 M Tris.HCI (pH 7.5)/0.05 M KC1/8% g l y c e r o l w e r e d i a l y z e d a t 4°C f o r 1 0 h a g a i n s t 1 0 0 m i o f t h e s a m e m e d i u m . T h e n t h e y w e r e i n c u b a t e d a t 37°C f o r 6 0 r a i n a n d f u r t h e r d i a l y z e d a t 4°C f o r 2 0 h a g , t n ~ t t h e s a m e m e d i u m c o n t a i n i n g 1 /~M d a u n o m y c i n . A f t e r dialysis, d a u n o m y c i n w a s e x t r a c t e d a n d d e t e r m i n e d as d e s c r i b e d i n t h e t e x t . Experiments of DNAase and pronase treatments were separate ones and different preparations of nonhistone proteins were used. Enzymatic treatment DNAase treatment Nonhistone proteins Nonhistone proteins Pronase treatment Nonhistone proteins Nonhistone proteins

Daunomycin bound (nmol/mg protein)

(0.162 mg) (0.126 mg) + DNAase* (0.2 mg)

3.05 (100) 3.16 (104)

( 0 . 0 5 3 rag) (0.053 rag) + Pronase

5.06 (100) 1.83 (36)

(0.2 mg)

* D N A a s e a c t i v i t y w a s s u c h t h a t w h e n 1 . 0 m l s o l u t i o n s o f D N A (6 # g ) in 0 . 0 1 M Tris*HC1 ( p H 7 . 5 ) w i t h and without DNAase (0.113 rag) were dialyzed against 100 ml of the same buffer plus 1/~M daunom y c i n a t 4°C f o r 2 0 h. A m o u n t s o f b o u n d d a u n o m y c i n w e r e 0 . 1 2 8 a n d 1 . 9 7 n m o L r e s p e c t i v e l y . T h e l a t t e r v a l u e is c o m p a r a b l e t o t h e l i t e r a t u r e [ 4 , 7 ] .

not be detected by the diphenylamine method [13]. Further, Table I shows that DNAase (Miles-Seravac Ltd., Maidenhead, U.K.) treatment of the proteins had no significant effect on the binding, indicating that DNA contamination, if any, may be negligible. It is known that DNAase-digested DNA will not interact with daunomycin [5]. On the other hand, preincubation in the presence of pronase E (Kaken Kagaku Co. Ltd., Tokyo) markedly decreased the extent of binding. The observed binding activity, therefore, may be attributed to a proteinaceous substance. The specificity of this binding was then examined. Table II shows that nonhistone proteins were by far the most potent in binding daunomycin among other proteins tested. It is evident, however, that the binding activity varied from preparation to preparation. Histones, in contrast, were very weak. Phosphoproteins, phosvitin prepared by the method of Mecham and Olcott [14] (kindly donated by Miss Kazuko Handa) and casein (Difco Laboratories, Detroit) were not specially potent but distinctly weak. Other non-phosphorylated proteins, catalase (Tokyo Kasei Industrial Co. Tokyo) T A B L E II DAUNOMYCIN

BINDING OF NONHISTONE

P R O T E I N S IN C O M P A R I S O N W I T H O T H E R P R O T E I N S

O n e - r a l s o l u t i o n s o f v a r i o u s p r o t e i n s i n 0 . 0 1 M TrisoHC1 ( p H 7 . 5 ) , 0 . 0 5 M KCI a n d 8% g l y c e r o l w e r e d i a l y z e d a g a i n s t 1 0 0 m l o f t h e s a m e m e d i u m p l u s 1 ~ M d a u n o m y e i n f o r 2 0 h a t 4°C. S i n c e t h e b i n d i n g activities of other proteins were weak compared with that of nonhistone, larger amounts of them were u s e d as i n d i c a t e d . A t t h e s e c o n c e n t r a t i o n s , n o o b v i o u s p r e c i p i t a t i o n w a s o b s e r v a b l e . Sample

A m o u n t o f p r o t e i n (rag)

Daunomycin bound (nmol/mg protein)

Nonhistone proteins Histones Phosvitin Casein Catalase Fibrlnogen

0.079 0. 5 0 0.50 1.00 0.50 0.50

4.45 + 1.62" 0.162 0.464 0.071 0.344 0.014

* M e a n +- S.D. o f 8 e x p e r i m e n t s .

512 and fibrinogen (Armour Pharmaceutical Company, Kankakee) were also weak binders. Thus, nonhistone proteins seem to be highly specific for binding to daunomycin. If daunomycin binds in vivo to nuclear nonhistone proteins associated with the region of the genome which codes for the synthesis of macromolecules essential for cell proliferation, the regulation, by the proteins, of gene expression [15--17] will be disturbed in the presence of the drug. If so, daunomycin will exert a dual effect on the cell proliferation, one at the regulatory level and the other at the level of DNA and RNA synthesis due to the widely known method of modifying the template activity of DNA. It will thus be interesting to determine the extent and relative importance of these two effects. References 1 2 3 4 5 6 7 S 9 10 11 12 13 14 15 16 17

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