A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 6, 1988
M a ł g o r z a t a Czyż, K a z i m i er z St ud zi an , A ndr z ej W a si le ws ki , A l e k s a n d r a P ozi o ms k a, D o r o t a W i lm a ńs k a, M a r e k G ni a z d ow s k i
C O V A L E N T B I N D I N G O F N I T R A C R I N E (LEDAKRIN, C-283) T O N U C L E I C A C I D S A N D P RO T EI N S
In the presence of sulfhydryl compounds an anticancer drug nitra- crine (NAC), l-nitro-9-(3, 3 N, N-dlmethylaminopropylamino) acridine binds irreversibly to DNA and RNA. The stability of the drug complex with DNA and RNA under various conditions were characterized. The fol-lowing facts indicate the drug in the purified complexes is covalently bound to nucleic acids: i) [*^C] NAC in the complex is precipitated with trichloacetic acid (TCA) with similar efficiency as DNA or RNA; ii) the complex Is not dissociated by 7 M urea, sodium dodecyl sulfa-te (SDS) or high ionic strength; iii) neither thermal nor alkaline denaturation considerably affect the efficiency of precipitation of [1AC] NAC-DNA with.TCA. In the presence of DTT [ 1AC] NAC binds ir-reversibly to both albumin and histone at. the level about tenfold lo-wer than that found for DNA.
INTRODUCTION
T h e a n t i c a n c e r d r u g n i t r a c r i n e (Ledakrin, C-28 3), NAC, 1-ni- t ro - 9- (3 , 3-N, N - d i m e t h y l a m i n o p r o p y l a m i n o ) a c r i d i n e u n d e r go e s m e -t a b o l i c a c -t i v a -t i o n in t he c e ll l e ad i ng to c o v a l e n t b i n d i n g to D NA a n d p r o b a b l y t o R N A a n d p r o t e i n s [1, 4, 8]. In the p r e s e n c e of s u l f h y d r y l c o m p o u n d s t he d r u g fo r ms w i t h D N A a nd R N A i r r e v er s ib l e c o m p l e x e s [2, 5]. T h e r e is a c o r r e l a t i o n b e t w e e n t he t hi o l - d e p en - d e n t b i n d i n g of s e v e r a l n i t r a c r i n e a n a l o g u e s to D N A a nd the ir b i o l o g i c a l e f f e c t s [5, 6, 10]. T h e a i m of t h e s e e x p e r im e n t s is to c h a r a c t e r i z e t he s t a b i l i t y of N A C b o u n d to m a cr o m o l e c ul e s .
MATERIALS
Alb umin , f r ac t io n V a c c or d i n g to Co hn and H ya m in e 2389 (Serva, He id el be rg , G F R ) , calf t hy mu s h i st o n H 1 k i nd l y d o n a t e d by Dr. Zo- fia M i l e w s k a f ro m our Inst itut e, w he a t g er m h ig h m o l e c u l a r w ei gh t R NA (C al bi och em , USA), G F/ C f il ter s (Whatman, E ngland), S ep ha de x G- 50 a nd G- 1 00 (Pharmacia, Sweden) a nd ca lf th ymu s D NA ( Wo rt h in -gton, USA) w e re used. 9 - [ 1 4C] N A C ( specific a c t i v i ty 3-6 . 1 0 3 cp m/n mo le ) w a s p r e v i o u s l y d e s c r i b e d [2].
METHODS
E s t i m a t i o n of s t o i c h i o m et r v of c o m p le x es of N AC w i t h n uc l e i c ac ids an d prot eins . If no t s ta t e d o th e rw i s e the c om p l ex e s w ere f or me d b y i n c ub a ti o n of n uc le i c aci ds or p r ot e i ns w i t h N A C in the p r e s en c e of 2 m M d i t h i ot h r e i t o l (DTT) for 1 h, p u r i f i e d b y e x -t r a c -t io n w i -t h i s ob u-t ano l and t he i r s t o i c h i o m e tr y e s t i m a t e d as d e -s c ri b ed b ef o re [2, 11] e x ce p t th at D NA a nd R NA c on c e n t r a ti o n s w e -re 400 vig/ml and hi st o ne ( H i ) c o n c e n t r a t i o n 2 mg/ml. K C1 or N aC l a n d N AC c o n c en t r a t i o ns are i n di c a te d in the d e s c r i p t i o n of e xp er im en ts. C om p l ex e s of a l b u m i n w e re f or me d as d e s c r i b e d above (a lbu mi n c o n c e n t r a t i o n w as 2 mg/m l) but p u r i fi e d b y f i lt r at i on t h r o ug h a S e p h ad e x G -50 c o l u m n (1.5 x 26 cm) p r e c i o u s l y e q u i l i -b r a t e d w i t h 0.05 M p ho s p ha t e buffer. P r ot e i n c o n c e n t r a ti o n wa s e s t i m a t e d a c c o r d in g to L o w r y [7]. A s s a y of s t a b i l i t y of c o m p l e x es of N A C w i t h n u c le i c acids. E xc e pt for s od i um d o de c y l s u lf a te (SDS) (see below) th e s t a b i l i -t y of c o m p l ex e s of N AC w i t h n u cl e i c acid s un d er d i f fe r e n t c o n d i -ti on s w as e s t i m a te d as follows: 0.2 m l a li qu o ts of the p ur i f ie d c o m p le x of N AC w i th D NA or R N A (250-4 00 yg/ml) w er e m a d e up to 1 ml b y the ad d it i o n of the c o r r e s p o n d i n g c o m po n e n t (20 m M Tris/ / H C 1 , pH 7.5, H C 1 , NaOH, urea) a nd i nc u ba t ed as spec ifie d, the n the s am pl e s a s s ay e d at l ow or h i g h p H w e re n eu tr aliz ed, T h e s a m -pl es w e r e p r e c i p i t a t e d w i t h 5 m l of 10% T C A at 0°C for 3 h, the p r e c i p i t a t e s w e r e c o l l e c t e d on G F /C filtres, w a s h e d 5-6 times w i t h c o ld 5% T C A t he n o nc e w i t h et hanol. T he fi lt er s w er e p la c ed in c o u n t i n g vials, t hen 0.5 ml of Hy amin e, 10% w as a dd e d to e ac h vial. T he sa mp les w e r e i n c u ba t ed for 24 h at 50°C, then 0.5 ml of m e t h a n o l an d 4 ml of t o lu e ne s ci n t i l l at o r - T r it o n
X- 1 00 (2 : 1) m i x t u r e w e r e ad d ed an d c o u nt e d as b ef o re [11]• Ea ch e x p e r i m e n t w a s d o ne in t ri p li ca te , a nd the r e su lt s are e x p r e ss e d as p e r c e n ta g e of the co nt ro ls , i.e. the u n t r e a t e d sa mp l es p r e c i p i -t a-t e d w i -t h T C A as above. E f f i c i e n c y of T C A p r e c i p i t a t i o n o f J ^ C ] N A C i r r e v e r s ib l y bo u nd to n uc l ei c aci ds wa s e s ti m a t e d fo l lo w i n g p r e c i p i t a t i o n of 0.2 ml a l i qu o ts of the p u r i f i e d c o m pl e x e s w i t h 10% T C A at 0 C for 3 h, w a s h i n g w i t h 5% T C A a nd eth an ol , s o l ub i l i z a t i o n w i t h H y am i ne and c o u n t i n g as d e s c r i b e d above. T he c on t ro l s w e r e the sa mp le s d i -re c t l y s p ot t e d and d r i e d o n t o G F/ C f il te rs p r e w a s h e d w i t h 5% T CA an d eth ano l. T he f il te rs w e r e p l a c e d in c o un t i n g vi al s an d s u -b j ec t e d to H y am i n e t r e a t m e n t a nd c o u n t e d (see above). T he e f f i -c i e n -c y of T C A p r e c i p i t a t i o n of D NA w a s e s t i m a t e d b y p h os p ho r us d e t e r m i n a t i o n [3] of t he y i e l d of D N A r e t ai n e d on G F/ C f ilte rs f ol l o wi n g T C A p r e c ip i ta t io n . S t a b i l i t y of [14C] N A C - D N A in the p r es e n c e of SDS wa s as sa ye d by i n c u b at i o n o f . 1 7 0- 2 10 yg of the c o m p l e x in 3 ml w i t h SDS (2.5%) an d N a C l (0.25 M) at 37°C for 3 h f o ll o we d b y f i l t ra t i o n th ro u gh S e p h a d e x G- 10 0 c o l u m n (1.5 x 26 cm) e q u i l i b r a t e d w i t h 0.05 M p h o s p h a t e bu ffer , pH 7.4. A b so r b a n c e at 260 n m an d the r a d i o -a c t i v i t y in the e l u -a t e w -as m e-a s ur ed . T he a mo u nt of r a d i o a c t i vi t y in th e m a i n p ea k c o r r e s p o n d i n g to the h i g h - m o l e c u l a r w e i g h t m a -t er i al w a s e x p r e s s e d as a p e r ce n t a g e of the t ot a l r ad i o a c t iv i t y r e c o v e r e d f r om the co lumn. A s s a y of s t a bi l i t y of c o m p l e x e s of N A C w i t h p r o t e i n s ^ Th e e f -f i c i e n c y o-f T C A p r e c i p i t a t i o n of N A C - a l b u m i n c o m p l e x w as fo l lo w ed by p r o t e i n d e t e r m i n a t i o n [7] a nd r a d i o a c t i v i t y m e a s u r e m e n t s un de r th e c o n d i t i o n s d e s c r i b e d for th e n u c le i c ac id s co mpl exes .
RESULTS AND DISCUSSION
T h i o l - d e p e n d e n t b i nd i n g of N A C to n uc l e i c acids._ In c ub a t i o n of N AC w i t h D N A in t he p r e s e n c e of s u l f h y dr y l c o m p o un d s leads to ti g ht i r r e ve r s i b l e b i n d i n g of the drug. G e n er a l p r o p e r t i e s of the r e a c t i o n m a y be s u m m a r i z e d as fol lows. T h e y i e l d of the add uct(s) f or m e d in the p r e s e n c e of D T T is c o n s i d e r a b l y h ig h e r t han that w i t h the ot h er t hi ols te sted: m e r c e p t o e t h a n o l [5, 9] or c y s te i ne ( u n p ub l is h ed o b se r va t io n ). As sh o wn o n Fig. 1 t he b i n d i n g of NAC d e p e n d s o n D TT c o n ce n t r a t i on , the o p t i m u m b ei n g o b s e r v e d at 2-5 mM. D e p e n d e n c e s of th e r e a c t i o n o n t im e an d on ioni c s tr e ng t h
[DDT] mM
[ N a C l ] mM (» 10' 2 )
Fig. 1. Effect of salt and DTT concentration on NAC binding to DNA
Complexes were formed by incubation of DNA (400 pg/ml) with [ ^ C ] NAC (0.1 mM in the presence of increasing concentration of NaCl (®) or DTT (•)
Rys. 1. Wpływ stężenia soli i DTT (ditiotreitolu) na wiązanie NAC z DNA Kompleksy uzyskano podczas inkubacji DNA (400 yg/ral) z [*4C] NAC (0,1 mM) w o-
becności wzrastającego stężenia NaCl (®) lub DTT (•)
a re s ho w n o n Figs. 1-2. T h e a d du c t f o rm a t i on w i t h n a t iv e a n d d e -n a t u r e d c alf th ymu s D N A a nd R N A d o e s n ot d i ff e r m o r e t ha n b y a f ac to r of t wo [2]. S t a b i l i t y of N A C c o m p l e xe s w i t h D N A a n d RNA. T he f ol l ow i ng fa cts i n d ic a te th at s u bs t a n t i al a m ou n t of the d r ug in the p u r i -f ie d c o m p l e x is a c t ua l l y c o v a l e n t l y b o u n d to n u cl e ic ac id B (Tab. 1): i) ov er 80% of the r a d i o a c t i v i t y is in s ol u bl e in T C A an d this a mo u nt is c lo se to the e f f i c i e n c y of D N A (90,7%) an d R N A (87.3%)
hours
Fig. 2. Time course of NAC binding to DNA
DNA concentration of 400 yg/ral with [ 1,4C ] NAC (0.097 mM) and DTT (2 mM) was incubated for the time indicated
Rys. 2. Wpływ czasu na wiązanie NAC z DNA
stężeniu 400 y g / m l było inkubowane z [*4C J NAC (0,097 mM) i DTT (2 mM) przez wskazany czas
p r e c i p i t a t i o n f o un d u n d er t he s e c on d it i on s ; ii) n e i th e r 7 M urea n or t h e r m a l o r a k a l i n e d é n a t u r a t i o n c o n s i d e r a b l y a ff e ct the e f -f i c i e n c y o-f p r e c i p i t a t i o n o-f [14C] N A C - D N A w i t h TCA; iii) 95. 5% o-f [ ^ c ] N A C f ro m th e c o m p l e x w i t h D N A m i g r a t e s w i t h h i g h m o l e c u l a r m a t e r i a l on S e p h a d e x c o l u m n af t er th e i n c ub a t i o n w i t h SDS and o v er 80% of b o t h t he l a b e ll e d d r u g a nd D N A f r om the c o m p l ex co- - p r e c i p i t a t e s w i t h SD S in the p r e s e n c e of 1 M N a C l (Tab. 1); iv) as s ho w n a l r e a d y [12] the N A C - D N A c o m p l e x is r es i s t an t to h i g h s a l t / p h e n o l t r eat me nt . D e c o m p o s i t i o n of the h i g h m o l e c u l ar m a t e r i a l is o b s e r v e d f o l lo w i n g h e a t i n g at pH 7.5 or pH 1.7 for 1 h. O t h e r p r o pe r t i e s of th e c o m p l e x e s are a l so s u m m a ri z e d in Tab. 1. T h e p r od u c t of th e r e a c t i o n of N A C w i t h R N A is s o m ew h at less s t ab l e d u r i n g t he i n c u b a t i o n at p H 7.5 at 37°C or u p o n h e a t i ng at 1 00 ° C (e.g. o n l y 66% is p r e c i p i t a t e d w i t h T C A f o l l o w i n g he-Native DNA at Natywne DNA w
T a b l e 1 Stability of NAC-DNA and NAC-RNA complexes
Stabilność kompleksów NAC-DNA i NAC-RNA
Conditions of treatment % of DNA control % of RNA control
TCA, 10%, 3 h, 0°C 87.4 + 4.9 (7)a 83.5 i 2.7 (2)a
TCA, 10%, 3 h, 0°C 90.7 + 5.5 (4 )b 87.3 i 5.1 (3)b SDS, 2.5%, 3 h, 37°C 95.5 4.9 (3)c SDS, 2.5%, 3 h, 37°C, then 0°C 83.5 + 4.0 (2)d -urea, 7 M, 1 h, 20°C 85.2 3.3 (5) 76.0 i 6.1 (4) pH 7.5, 70 h, 37°C 81.4 + 1.6 (2) . 75.3 t 0.9 (2) pH 7.5, 10 min, 100°C 89.0 + 3.0 (2) 65.9 i 0.6 (2) pH 7.5, 1 h, 100°C 57.8 t 9.7 (2) 50.3 i 1.5 (2) NaOH, 0.01 M, 1 h, 20°C 98.2 t 2.9 (3) 96.6 t 0.7 (2) NaOH, 0.05 M, I h, 20°C 95.7 + 1.1 (3) 93.9 i 0.3 (2) NaOH, 0.5 M, 18 h, 30°C - 32.6 ± 1.3 (4) pH 1.7, 1 h, 100°C 22.4 6.1 (3) 37.2 ± 5.8 (5)
a ’ ^ Efficiency of precipitation of NAC-DNA and NAC-RNA complexes with TCA estimated by radioactivity measurements (a) and phosphorus determination (b).
c NAC-DNA complex following treatment with SDS was analyzed on Sephadex G- -100 column.
d NAC-DNA treated as (c) but in the presence of 1 M NaCl instead of 0.25 M NaCl then cooled to 0°C. The material co-precipitated with SDS contained over 80% of DNA and indicated amount of radioactivity.
N o t e : In all the experiments except these denoted with superscripts a-d the samples of NAC-DNA (or NAC-RNA) were treated as indicated then precipita-ted with TCA and counprecipita-ted. The results are expressed as percentage of the cor-responding controls, i.e. the untreated NAC-DNA or NAC-RNA complex. Averages of two or more experiments are given (as indicated in the parantheses) ± range or standard deviations values.
at i ng in a b o il i ng wa t e r ba t h for 10 m i n . ; see Tab. 1). Ra t h e r s u r -p r i s i n g l y [14C] N A C - R N A is r e s is t an t in the h i g h l y al k al i n e m e -dium. As m u c h as 33% of the r a d i oa c t i v i t y re ma in s a ci d i ns o lu bl e un der the c o n d it i o n s w h i c h are u se d to hy d ro l yz e R NA (in cu b at i on w i t h 0.5 M N a OH for 18 h). S i m il a r a mo un t of the d ru g is o b s e r -v ed at the o r i g i n of the pa p er st ri p w h e n the a l k al i ne h y d r o l y s a -te of N A C - R N A is s ub j ec t e d to c h r o m a t o g r a p h y in e t h an o l / a m m o n i u m a c et a te ( 7 : 3 ) w h i l e the rest of r a d io a c t i v i t y m i gr a t e s w i t h n u -c l eo t i d e s in two peaks. Th e y i e l d of ph o s ph o r u s R N A p r e c i pi t a t e d w i t h T C A f o l l o wi n g i n c u b a ti o n w i t h 0.5 M N aC l is h o we v er simil ar
w i t h i n t he a c c u r a c y of the d e t e r m i n a t i o n for N A C - R N A arv* free RN A (not s h o w n ) . N A C b i n d i n g to p rot ei ns . T h e a m ou n t of the d r u g w h i c h is c o -p u r i f i e d w i t h h i s t o n e H 1 or w i t h a l b u m i n f o l l o w i ng i n c ub a ti o n of 1 4 p r o t e i n w i t h [ C] N A C the p r e s e n c e of D T T is c o n s i d e r a b ly h ig h er t ha n th at f ou n d in p r o t e i n i n c ub a t e d w i t h o u t s u lf h yd r yl c o m p o u n d s (Tab. 2). A l t h o u g h the c o n d i t i o ns of the r e a c t i o n w it h
T a b l e 2 Binding of NAC to proteins
Wiązanie NAC z białkami
Specification [U C ] NAC mM nmole/mg I Histone HI 0.13 5.1 II Histone HI 0.20 9.2 Histone HI - DTT 0.20 1.4a DNA - DTT 0.20 0. 4b III Albumin 0.14 6.6 IV Albumin 0.14 9.0 Albumin - DTTa 0.14 0.4
a The control without DTT.
b DNA incubated with [U C ] NAC but without DTT. The amount of [1AC] NAC bound to DNA in the presence of DTT is about 50 nmole/mg under these condi-tions.
N o t e : Concentration of [ ^ C ] NAC (mM) during the complex formation and the amount of the drug bound to protein (nmole/mg) is indicated.
p r o t e i n s a re s o m e w ha t d i f f e r e n t t ha n t ho se u se d for D N A the a p -p r o x i m a t i v e le ve l of b i n d i n g of N A C to p r o t e i n s pe r m a ss u ni t is a b ou t t e n f o l d lo we r t h an t h at f ou nd for DNA. It is s im il a r for the tw o p r o t e i n s as d i f f e r e n t as h is t o n e an d a l b u m i n (Tab. 2). A l t h o u g h th e p r o t e i n - d r u g c o m p l e x e s w er e less t h o r o u g h l y s t u -d i e -d t h an t h os e w i t h n u c l e i c a ci d s f o l l o w i n g fa ct s i nd i ca t e that c o v a l e n t b i n d i n g d oe s occur: i) t he r e is no r e l e a se of the label f r om th e p u r i f i e d [1 4C] N AC a l b u m i n c o m p l e x w h ç n it is i n cu b at e d w i t h an e x c e s s of the u n l a b e l l e d d r u g - 9 7- 99 % of the r a d i o a c t i -v i t y is e l u t e d w i t h t h e p r o t e i n p e a k f r om S e p h a d e x G -50 column; ii) o ve r 90% of the r a d i o a c t i v i t y is c o - e l u t e d w i t h p r o t e i n p ea k f o l l o w i n g i n c u b a t i o n of th e c o m p l e x w i t h SDS.
ACKNOWLEDGEMENTS
Th e au th o rs tha nk Dr. W. A. D e n n y for re ad i ng a dr a f t of this m a n u s c r i p t a nd h e lp f ul c om m en t s and to M is s M. A f f el t ow i cz for t e c h ni c al ass ist an ce. T hi s w o r k is s up p or t e d b y g ra n t No 2304 of the P ol i s h N a t io n a l C an cer Program.
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Department of General Chemistry Institute of Physiology and Biochemistry Medical Academy of Łódź
Małgorzata Czyi, Kazimierz Studzian, Andrzej Wasilewski, Aleksandra Poziomska, Dorota Wilmańska, Marek Gniazdowski
WIĄZANIE KOWALENCYJNE NITRAKRYNY (LEDAKRIN, C-283) Z KWASAMI NUKLEINOWYMI I BIAŁKAMI
W obecności związków sulfhydrylowych lek przećiwnowot.worowy nitrakryna (Le- dakrin, C-283) tworzy nieodwracalne kompleksy z DNA i RNA. Przedstawiono cha-rakterystyką stabilności kompleksów w różnych warunkach. Szereg faktów wskazu-je na kowalencyjny charakter wiązania pomiędzy Ledakrinem a kwasami nukleino-wymi: 1) [ 14C] NAC znajdujący się w oczyszczonym kompleksie jest wytrącany kwasem trój chlorooctowym z podobną wydajnością jak DNA czy RNA; 2) kompleksy nie dysocjują w roztworach 7 M mocznika i siarczanu dodecylu sodu (SDS) oraz w wysokiej sile jonowej; 3) zarówno termiczna, jak i alkaliczna denaturacja nie wpływa znacząco na wydajność strącania kompleksu [1/łC J NACDNA przy p o -mocy kwasu trój chlorooctowego. Ponadto stwierdzono, że w obecności ditiotrei- tolu (DTT) [U C ] NAC wiąże się w sposób nieodwracalny z albuminą i histonem, chociaż ilość związanego leku jest dziesięciokrotnie niższa niż w przypadku DNA.
